Mixotrophic Chlorella-Based Composition, and Methods of its Preparation and Application to Plants

ABSTRACT

Methods preparing a liquid mixotrophic  Chlorella  based composition comprising pasteurization and stabilization of a low concentration of mixotrophic  Chlorella  whole cells are disclosed. The liquid composition can be used to enhance the emergence and growth of plants in low concentration and low frequency soil, foliar, seed soak, and hydroponic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims benefit to U.S.application Ser. No. 15/536,705 titled Mixotrophic Chlorella-BasedComposition, and Methods of its Preparation and Application to Plantsand filed on Jun. 16, 2017 in the name of Applicant, wherein U.S.application Ser. No. 15/536,705 in turn claims the benefit of PCTApplication No. PCT/US2015/066160 titled Mixotrophic Chlorella-BasedComposition, and Methods of its Preparation and Application to Plantsand filed on Dec. 16, 2015 in the name of the Applicant and wherein PCTApplication No. PCT/US2015/066160 claims the benefit of U.S. ProvisionalApplication No. 62/092,766, filed Dec. 16, 2014, titled Methods ofPreparing a Mixotrophic Chlorella Based Composition for Application toPlants; U.S. Provisional Application No. 62/092,771, filed Dec. 16,2014, titled Application of Mixotrophic Chlorella for the AcceleratedEmergence and Maturation of Fabaceae Plants; U.S. ProvisionalApplication No. 62/092,774, filed Dec. 16, 2014, titled Application ofMixotrophic Chlorella for the Accelerated Emergence and Maturation ofSolanaceae Plants; U.S. Provisional Application No. 62/092,703, filedDec. 16, 2014, titled Application of Mixotrophic Chlorella for theImproved Yield and Quality of Solanaceae Plants; U.S. ProvisionalApplication No. 62/092,777, filed Dec. 16, 2014, titled MixotrophicChlorella Based Composition for Application to Plants; U.S. applicationSer. No. 14/602,331, filed Jan. 22, 2015, titled Methods of Preparing aMixotrophic Chlorella Based Composition for Application to Plants; U.S.application Ser. No. 14/602,348, filed Jan. 22, 2015, titled Applicationof Mixotrophic Chlorella for the Accelerated Emergence and Maturation ofFabaceae Plants; U.S. application Ser. No. 14/602,356, filed Jan. 22,2015, titled Application of Mixotrophic Chlorella for the AcceleratedEmergence and Maturation of Solanaceae Plants; and U.S. application Ser.No. 14/602,362, filed Jan. 22, 2015, titled Application of MixotrophicChlorella for the Improved Yield and Quality of Solanaceae Plants. Theentire contents of all of the foregoing are hereby incorporated byreference herein.

BACKGROUND

Seed emergence occurs as an immature plant breaks out of its seed coat,typically followed by the rising of a stem out of the soil. The firstleaves that appear on many seedlings are the so-called seed leaves, orcotyledons, which often bear little resemblance to the later leaves.Shortly after the first true leaves, which are more or less typical ofthe plant, appear, the cotyledons will drop off. Germination of seeds isa complex physiological process triggered by imbibition of water afterpossible dormancy mechanisms have been released by appropriate triggers.Under favorable conditions rapid expansion growth of the embryoculminates in rupture of the covering layers and emergence of theradicle. A number of agents have been proposed as modulators of seedemergence. Temperature and moisture modulation are common methods ofaffecting seed emergence. Addition of nutrients to the soil has alsobeen proposed to promote emergence of seeds of certain plants.

Similarly, the growth and fruit production of a mature plant is also acomplex physiological process involving inputs and pathways in theroots, shoot, and leaves. Whether at a commercial or home garden scale,growers are constantly striving to optimize the yield and quality of acrop to ensure a high return on the investment made in every growthseason. As the population increases and the demand for raw plantmaterials goes up for the food and renewable technologies markets, theimportance of efficient agricultural production intensifies. Theinfluence of the environment on a plant's health and production hasresulted in a need for strategies during the growth season which allowthe plants to compensate for the influence of the environment andmaximize production. Addition of nutrients to the soil or application tothe foliage has also been proposed to promote yield and quality incertain plants. The effectiveness can be attributable to the ingredientsor the method of preparing the product. Increasing the effectiveness ofa product can reduce the amount of the product needed and increaseefficiency of the agricultural process.

SUMMARY

Embodiments of the present invention provide methods for preparing aliquid mixotrophic Chlorella based composition. The composition caninclude pasteurization and stabilization of a low concentration ofmixotrophic Chlorella whole cells that have not been subjected to adrying process. The liquid composition can be used to enhance theemergence and growth of plants in low concentration and low frequencysoil and foliar applications.

Some embodiments include a method of plant enhancement which includesadministering to a plant a liquid composition treatment which includes aculture of Chlorella. The composition can include whole pasteurizedChlorella cells. In some embodiments, the composition can beadministered in a concentration in the range of 0.003-0.080% solids byweight.

In some embodiments, the Chlorella cells can be pasteurized at between50 and 80° C. for a time between 15 and 360 minutes. In someembodiments, the Chlorella cells can be pasteurized in a culture havinga concentration greater than 11% by weight of Chlorella, at between 55and 65° C. for between 90 and 150 minutes. In some embodiments, theculture can be then diluted to 10-11% Chlorella by weight and cooled tobetween 35 and 45° C. In some embodiments, the pasteurized culture canadjusted to a pH between 3.5 and 4.5.

In some embodiments, the Chlorella cells can be cultured in mixotrophicconditions. In some embodiments, the mixotrophic conditions includeculturing the Chlorella cells in a suitable medium for a culture lengthof 7-14 days, at a temperature between 20 and 30° C., at a pH between6.5 and 8.5, and a dissolved oxygen concentration can range between 0.1and 4 mg/L.

In some embodiments, the Chlorella cells can be cultured in non-axenicmixotrophic conditions. In some embodiments, at least one species ofsporulating bacterium can be present in the non-axenic culture. Thebacterium can be Paenibacillus sp., Bacillus sp., Lactobacillus sp.,Brevibacillus sp., or similar.

In some embodiments, administration of the composition can be by soakinga seed in the composition prior to planting; contacting soil in animmediate vicinity of a planted seed with an effective amount of thecomposition; contacting roots of a plant with an effective amount of thecomposition hydroponically; contacting an effective amount of thecomposition to an accessible portion of the plant after emergence; orsimilar.

In some embodiments, the liquid composition can be administered at arate in the range of 10-150 gallons per acre to soil or to emergedplants in soil.

In some embodiments, the seed can be soaked for 90-150 minutes.

In some embodiments, the liquid composition can include 0.008-0.080%solids by weight of whole pasteurized Chlorella cells.

In some embodiments, the liquid compositions can be administered byspraying. The compositions can be administered every 3-28 days or every4-10 days or similar. In some embodiments, the liquid composition can befirst administered 5-14 days after emergence.

In some embodiments, the liquid composition can be administered to thesoil by a low volume irrigation system, a soil drench application, anaerial spraying system, or the like.

In some embodiments, the plant can be a member of a plant familySolanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae, Vitaceae,Brassicaeae (Cruciferae), Caricaceae, Malvaceae, Sapindaceae,Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae,Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae(Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae,Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae,Rubiaceae, Papveraceae, Illiciaceae Grossulariaceae, Myrtaceae,Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae),Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae,Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae,Piperaceae, Proteaceae, or the like.

In some embodiments, the whole Chlorella cells have not been subjectedto a drying process.

In some embodiments, the liquid composition treatment can furtherinclude at least one culture stabilizer suitable for plants. The culturestabilizer can be potassium sorbate, phosphoric acid, ascorbic acid,sodium benzoate, or the like, or any combination thereof.

In some embodiments, the liquid composition treatment does not containan active ingredient for enhancing emergence or maturation other thanthe culture of whole Chlorella cells.

In some embodiments, enhancement can be determined by comparison of atreated plant with a substantially identical untreated plant. Aquantifiable difference of at least 10% can be observed for at least oneplant characteristic.

In some embodiments, the plant characteristic can be seed germinationrate, seed germination time, seedling emergence, seedling emergencetime, seedling size, plant fresh weight, plant dry weight, utilization,fruit production, leaf production, leaf formation, thatch height, planthealth, plant resistance to salt stress, plant resistance to heatstress, plant resistance to heavy-metal stress, plant resistance todrought, maturation time, yield, root length, root mass, color, insectdamage, blossom end rot, softness, fruit quality, and sunburn., or thelike, or any combination thereof.

In some embodiments, the number of plants emerged from the soil can beincreased by at least 10% compared to a substantially identicalpopulation of untreated plants. In some embodiments, the number ofplants demonstrating maturation by leaf formation can increased by atleast 10% compared to a substantially identical population of untreatedplants.

Embodiments of the invention provide a liquid composition for plantenhancement, the composition including whole pasteurized Chlorellacells. In some embodiments, the composition can be a pasteurized axenicculture, wherein at least one species of sporulating bacterium can bepresent. In some embodiments, the bacterium can be selected fromPaenibacillus sp., Bacillus sp., Lactobacillus sp., Brevibacillus sp.,and any combination thereof. In some embodiments, the bacterial floraincludes at least five other bacteria in addition to all ofPaenibacillus sp., Bacillus sp., Lactobacillus sp., Brevibacillus sp.The bacterial flora can also include any one or more of the bacterialisted below:

Paenibacillus sp. Bacillus sp. Lactobacillus sp. Brevibacillus sp.Massilia sp. Pseudomonas sp.

Bdellovibrio bacteriovorus

Stenotrophomonas sp. Acinetobacter sp. Enterobacter sp. Flavobacteriumsp.

Zoogloea sp. Algae associated

Burkholderiaceae—fam Xanthomonadaceae—fam Enterobacteriaceae—famComamonadaceae—fam Oxalobacteraceae—fam Chitinopagaceae—fam

Gamma-proteobacterium—class

Burkholderiales—fam Proteobacteria—phy Singleton Taxa

Unclassified bacterium

In some embodiments of the invention, the composition can be from0.0001%-40% Chlorella by weight. In some embodiments, the can be 10-11%Chlorella by weight. In some embodiments, the pasteurized culture can beat a pH between 3.5 and 4.5. In various embodiments, the Chlorella canbe a mixotrophic culture, grown on at least one organic carbon source aswell as via photosynthesis. In some embodiments, the composition furtherincludes a suitable medium for Chlorella growth. In some embodiments,the composition can be diluted with water. Likewise, in someembodiments, the composition can include other additives includingfertilizers, pH adjusters, plant hormones, insecticides, minerals,detergents. In some embodiments, the composition does not contain anadditive for enhancing emergence or maturation other than the culture ofwhole Chlorella cells.

Some embodiments of the invention provide a soil harboring a plant seed,including the composition, wherein the seed has an improvedcharacteristic. In some embodiments, the invention provides a plantseed, contacted by the composition, wherein the seed has an improvedcharacteristic. Likewise, in some embodiments, the invention providesroots of a plant contacted with an effective amount of the composition,hydroponically or in soil, wherein the root or the whole plant has animproved characteristic. In still other embodiments, the inventionprovides a plant contacted with the composition after emergence, whereinthe plant has an improved characteristic.

Some embodiments of the invention include a low volume irrigation systemincluding the composition. In some embodiments, the invention includes asoil drench system, and/or an aerial spraying system including thecomposition.

In some embodiments, the composition further includes at least oneculture stabilizer suitable for plants. In some embodiments, the culturestabilizer can be selected from: potassium sorbate, phosphoric acid,ascorbic acid, sodium benzoate, and any combination thereof.

Some embodiments of the invention provide harvested material from aplant contacted by the composition, wherein the plant displayed animproved characteristic. In some embodiments, the improvedcharacteristic can be present in the harvested material.

Some embodiments of the invention include turf contacted by thecomposition, the turf having an improved characteristic. In someembodiments, a field of such turf can be provided as part of theinvention. In some embodiments, the invention includes a golf coursewith the turf.

Some embodiments of the invention provide a plant renderedstress-resistant by contact with the composition. Likewise, someembodiments of the invention provide soil rendered arable by spraying orsoaking with the composition in presence of seeds or plants madesufficiently resistant to stress to be capable of growth in the soil.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of a test for the condition of the DNA ofChlorella cells after pasteurization (DAPI).

FIG. 2 depicts the results of a test for the condition of cell membraneof Chlorella cells after pasteurization (propidium iodine).

FIG. 3 depicts the results of various pasteurization conditions of thecomposition.

FIG. 4 depicts the results of various application rates of thecomposition.

FIG. 5A-5F depicts graphs of the results of the composition onsalt-stressed plants.

FIG. 6A-6B depicts results of experiments using the composition on turf.

FIG. 7A-7B depicts results of experiments using the composition onpeanuts.

FIG. 8 depicts results of experiments involving different preparationsof the composition.

DETAILED DESCRIPTION

Many plants can benefit from the application of liquid compositions thatprovide a bio-stimulatory effect. Non-limiting examples of plantfamilies that can benefit from such compositions can include:Solanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae, Vitaceae,Brassicaeae (Cruciferae), Caricaceae, Malvaceae, Sapindaceae,Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae,Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae(Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae,Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae,Rubiaceae, Papveraceae, Illiciaceae Grossulariaceae, Myrtaceae,Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae),Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae,Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae,Piperaceae, and Proteaceae.

The Fabaceae plant family (also known as the Leguminosae) comprises thethird largest plant family with over 18,000 species, including a numberof important agricultural and food plants. Taxonomically classified inthe Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta(superdivision), Magnoliophyta (division), Manoliopsida (class), Rosidae(subclass), and Fabales (order), the Fabaceae family includes, but isnot limited to, soybeans, beans, green beans, peas, chickpeas, alfalfa,peanuts, sweet peas, carob, and liquorice. Plants in the Fabaceae familycan range in size and type, including but not limited to, trees, smallannual herbs, shrubs, and vines, and typically develop legumes. Plantsin the Fabaceae family can be found on all the continents, excludingAntarctica, and thus have a widespread importance in agriculture acrossthe globe. Besides food, plants in the Fabaceae family can be used toproduce natural gums, dyes, and ornamentals.

The Solanaceae plant family includes a large number of agriculturalcrops, medicinal plants, spices, and ornamentals in its over 2,500species. Taxonomically classified in the Plantae kingdom, Tracheobionta(subkingdom), Spermatophyta (superdivision), Magnoliophyta (division),Manoliopsida (class), Asteridae (subclass), and Solanales (order), theSolanaceae family includes, but is not limited to, potatoes, tomatoes,eggplants, various peppers, tobacco, and petunias. Plants in theSolanaceae can be found on all the continents, excluding Antarctica, andthus have a widespread importance in agriculture across the globe.

The Poaceae plant family supplies food, building materials, andfeedstock for fuel processing. Taxonomically classified in the Plantaekingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision),Magnoliophyta (division), Liliopsida (class), Commelinidae (subclass),and Cyperales (order), the Poaceae family includes, but is not limitedto, flowering plants, grasses, and cereal crops such as barely, corn,lemongrass, millet, oat, rye, rice, wheat, sugarcane, and sorghum. Typesof turf grass found in Arizona include, but are not limited to, hybridBermuda grasses (e.g., 328 tifgrn, 419 tifway, tif sport).

The Rosaceae plant family includes flowering plants, herbs, shrubs, andtrees. Taxonomically classified in the Plantae kingdom, Tracheobionta(subkingdom), Spermatophyta (superdivision), Magnoliophyta (division),Magnoliopsida (class), Rosidae (subclass), and Rosales (order), theRosaceae family includes, but is not limited to, almond, apple, apricot,blackberry, cherry, nectarine, peach, plum, raspberry, strawberry, andquince.

The Vitaceae plant family includes flowering plants and vines.Taxonomically classified in the Plantae kingdom, Tracheobionta(subkingdom), Spermatophyta (superdivision), Magnoliophyta (division),Magnoliopsida (class), Rosidae (subclass), and Rhammales (order), theVitaceae family includes, but is not limited to, grapes.

Particularly important to plant production is the beginning stage ofgrowth where the plant emerges and matures into establishment. A methodof treating a seed, seedling, or plant to directly improve thegermination, emergence, and maturation of the plant; or to indirectlyenhance the microbial soil community surrounding the seed or seedling istherefore valuable in starting the plant on the path to marketableproduction. The standard typically used for assessing emergence is theachievement of the hypocotyl stage, where a stem is visibly protrudingfrom the soil. The standard typically used for assessing maturation isthe achievement of the cotyledon stage, where two leaves visibly form onthe emerged stem.

Also important in the production of fruit from plants is the yield andquality of fruit, which can be expressed in terms of, for example, thenumber, weight, color, firmness, ripeness, moisture, degree of insectinfestation, degree of disease or rot, and/or degree of sunburn of thefruit. A method of treating a plant to directly improve thecharacteristics of the plant, or to indirectly enhance the biochemistryof the plant for photosynthetic capabilities and health of the plant'sleaves, roots, and shoot to enable robust production of fruit istherefore valuable in increasing the efficiency of marketableproduction. Marketable and unmarketable designations can apply to boththe plant and fruit, and can be defined differently based on the end useof the product, such as but not limited to, fresh market produce andprocessing for inclusion as an ingredient in a composition. Themarketable determination can assess such qualities as, but not limitedto, color, insect damage, blossom end rot, softness, and sunburn. Theterm total production can incorporate both marketable and unmarketableplants and fruit. The ratio of marketable plants or fruit tounmarketable plants or fruit can be referred to as utilization andexpressed as a percentage. The utilization can be used as an indicatorof the efficiency of the agricultural process as it shows the successfulproduction of marketable plants or fruit, which will obtain the highestfinancial return for the grower, whereas total production will notnecessarily provide such an indication. In addition, improvements in andmeasures of plant health can include plant resistance to stress.Stresses can be abiotic, such as, for example, temperature stress (hightemperature as well as frost), salt stress, heavy-metal stress, waterstress (whether drought or overwatering), and the like. Likewise,stresses can be biotic, such as, for example, stresses caused by fungi,bacteria, insects, weeds, viruses, and the like. Measures of improvedplant health can be qualitative or quantitative. When quantitative,embodiments of improvement in plant health can be a relative improvementin any characteristic as compared to an untreated plant, wherein theimprovement is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%,750%, 1000%, or more.

To achieve such improvements in health, emergence, maturation, yield,and quality of plants, embodiments of the invention provide microalgaebased compositions, methods of preparing liquid microalgae basedcompositions, and methods of applying the microalgae based compositionsto plants. The microalgae of the liquid composition can compriseChlorella sp. cultured in mixotrophic conditions, which comprises aculture medium primarily comprised of water with trace nutrients (e.g.,nitrates, phosphates, vitamins, metals found in BG-11 recipe (availablefrom UTEX The Culture Collection of Algae at the University of Texas atAustin, Austin, Tex.)), light as an energy source for photosynthesis,organic carbon (e.g., acetate, acetic acid) as both an energy source anda source of carbon. In some embodiments, the Chlorella can be culturedin non-axenic mixotrophic conditions in the presence of contaminatingorganisms, such as but not limited to bacteria. Methods of culturingsuch microalgae in non-axenic mixotrophic conditions can be found inWO2014/074769A2 (Ganuza, et al.), hereby incorporated by reference.

In one non-limiting example of mixotrophic culturing of Chlorella forthe described method of preparation of a composition for application toplants, the Chlorella is cultured in a BG-11 culture media or mediaderived from BG-11 culture media (e.g., in which additional component(s)are added to the media and/or one or more elements of the media isincreased by 5%, 10%, 15%, 20%, 25%, 33%, 50%, or more over unmodifiedBG-11 media) for a culture length of 7-14 days in an open culturingvessel. The temperature can range from 20−30° C. and the pH from6.5-8.5. The dissolved oxygen concentration can range from 0.1-4 mg/L.The culture receives acetic acid or acetate as a source of organiccarbon supplying both carbon and an energy source to the Chlorellacells, and is supplied to the culture in a feed with a concentration inthe range of 10-90% by a pH auxostat system. The culture receivesnatural sunlight (comprising photosynthetically active radiation) assource of energy. Mixing is provided by air sparging through aerotube,and fluid propulsion by thrusters submerged in the liquid culture.

By artificially controlling aspects of the Chlorella culturing processsuch as the organic carbon feed, oxygen levels, pH, and light, theculturing process differs from the culturing process that Chlorellaexperiences in nature. In addition to controlling various aspects of theculturing process, intervention by human operators or automated systemsoccurs during the non-axenic mixotrophic culturing of Chlorella throughcontamination control methods to prevent the Chlorella from beingoverrun and outcompeted by contaminating organisms (e.g., fungi,bacteria). Contamination control methods for microalgae cultures areknown in the art and such suitable contamination control methods fornon-axenic mixotrophic microalgae cultures are disclosed inWO2014/074769A2 (Ganuza, et al.), hereby incorporated by reference. Byintervening in the microalgae culturing process, the impact of thecontaminating microorganisms can be mitigated by suppressing theproliferation of containing organism populations and the effect on themicroalgal cells (e.g., lysing, infection, death, clumping). Thusthrough artificial control of aspects of the culturing process andintervening in the culturing process with contamination control methods,the Chlorella culture produced as a whole and used in the describedinventive compositions differs from the culture that results from aChlorella culturing process that occurs in nature.

In the alternative, the method of culturing Chlorella mixotrophicallycan comprise other known sources of organic carbon or combinations oforganic carbon sources, such as: ammonium linoleate, arabinose,arginine, aspartic acid, butyric acid, cellulose, citric acid, ethanol,fructose, fatty acids, galactose, glucose, glycerol, glycine, lacticacid, lactose, maleic acid, maltose, mannose, methanol, molasses,peptone, plant based hydrolyzate, proline, propionic acid, ribose,sacchrose, partial or complete hydrolysates of starch, sucrose,tartaric, TCA-cycle organic acids, thin stillage, urea, industrial wastesolutions, and yeast extract; as well as other known methods of mixing,methods of organic carbon supply, lighting, culture media, nutrientstocks, culturing vessels, and optimization of the culture parameterssuch as but not limited to temperature, pH, dissolved oxygen, anddissolved carbon dioxide. The mixotrophic Chlorella culture can beharvested from the culturing vessel and/or concentrated by means knownin the art, such as but not limited to, settling, centrifugation,filtration, and electrodewatering to form the mixotrophic Chlorellabased composition that is used in the final product composition.

During the mixotrophic culturing process the Chlorella culture can alsocomprise cell debris and compounds excreted from the Chlorella cellsinto the culture medium. The output of the Chlorella mixotrophicculturing process provides the active ingredient for a composition thatis applied to plants for improving at least one plant performancecharacteristic such as, for example, emergence, maturation, yield,quality, and the like. Typically, the composition is applied withoutseparate addition to or supplementation of the composition with otheractive ingredients not found in the mixotrophic Chlorella whole cellsand accompanying culture medium from the mixotrophic culturing processsuch as, but not limited to: non-Chlorella microalgae cells, microalgaeextracts, macroalgae, macroalgae extracts, liquid fertilizers, granularfertilizers, mineral complexes (e.g., calcium, sodium, zinc, manganese,cobalt, silicon), fungi, bacteria, nematodes, protozoa, digestatesolids, chemicals (e.g., ethanolamine, borax, boric acid), humic acid,nitrogen and nitrogen derivatives, phosphorus rock, pesticides,herbicides, insecticides, enzymes, plant fiber (e.g., coconut fiber);however, in some embodiments the augmentation of the base compositionwith any of the foregoing is contemplated. In the alternative, themixotrophic Chlorella based composition can be supplemented withnitrogen, phosphorus, or potassium to increase the levels within thecomposition to at least 1% of the total composition (i.e., addition ofN, P, or K to increase levels at least 1-0-0, 0-1-0, 0-0-1, orcombinations thereof). In some embodiments, the supplemented nutrient isnot uptaken, chelated, or absorbed by the microalgae.

Mixotrophic Chlorella is the dominant microalgae species in the liquidcomposition. In some embodiments, the microalgae population of theliquid composition is substantially mixotrophic Chlorella. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least90% of the microalgae population of the liquid composition. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least91% of the microalgae population of the liquid composition. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least92% of the microalgae population of the liquid composition. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least93% of the microalgae population of the liquid composition. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least94% of the microalgae population of the liquid composition. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least95% of the microalgae population of the liquid composition. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least96% of the microalgae population of the liquid composition. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least97% of the microalgae population of the liquid composition. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least98% of the microalgae population of the liquid composition. In someembodiments, mixotrophic or non-mixotrophic Chlorella comprises at least99% of the microalgae population of the liquid composition. Liquidcompositions having at least 99% of a Chlorella microalgae strain (e.g.,at least 99.3%, at least 99.5%, or even at least 99.9%), such asmixotrophic Chlorella, can be considered to have a single algal speciesin the liquid composition. In one aspect, the liquid composition lacksany detectable amount of any other microalgae species. In anotheraspect, the liquid composition lacks any amount of any othermicroorganism (e.g., bacteria) in the liquid composition other than thedesired Chlorella microalgae that is above 1% of the composition byweight.

The mixotrophic Chlorella resulting from the culturing stage consists ofwhole cells with the proximate analysis shown in Table 1, fatty acidprofile shown in Table 2, and results of further analysis shown inExamples 1-3. The nutrient profile (i.e. proximate analysis) of themixotrophic Chlorella cells before and after pasteurization, as well asduring subsequent storage, was found to have little variance in mostembodiments.

TABLE 1 Range Moisture & Volatiles 1-2% Ash Content   3-4.5%Carbohydrates 30-36% (calculated) % Protein (Leco) 15-45% % Lipids(AOAC)  5-20%

TABLE 2 Analyte Range (%) C16 Palmitic Acid 0.1-4 C18:1n9c Oleic acid(Omega-9) 0.1-2 C18:2n6c Linoleic acid (Omega-6) 0.1-5 C18:3n3Alpha-Linoleic acid (Omega-3) 0.1-2 Other 0.1-4 Total  0.5-17

The mixotrophic Chlorella cells can also contain detectable levels ofphytohormones, such as but not limited to: abscisic acid andmetabolites, which are known to be related to the stomal apparatusfunction, growth inhibition, and seed dormancy; cytokinins, which areknown to be related to cell division, bud development, development ofthe leaf blade, and senescence retardation; auxins, which are known tobe related to elongation growth, differentiation of phloem elements,apical dominance, tropism, and initial root formation; and gibberellins,which are known to be related to stem elongation and initiation of seedgermination.

In some embodiments, the mixotrophic Chlorella can comprise abscisicacid and abscisic acid metabolites in a range of 5-45 ng/g dry weight(DW). In some embodiments, the mixotrophic Chlorella can comprisecytokinins in a range of 60-300 ng/g dry weight (DW). In someembodiments, the mixotrophic Chlorella can comprise auxins in a range of400-815 ng/g dry weight (DW). In some embodiments, the mixotrophicChlorella can comprise gibberellins in a range of 0.1-15 ng/g dry weight(DW). In some embodiments, the mixotrophic Chlorella can comprisespecific phytohormones in the ranges shown in Table 3.

In some embodiments, the mixotrophic Chlorella can comprise abscisicacid and abscisic acid metabolites in a range of 0.1-1 ng/g fresh weight(FW). In some embodiments, the mixotrophic Chlorella can comprisecytokinins in a range of 10-30 ng/g fresh weight (FW). In someembodiments, the mixotrophic Chlorella can comprise auxins in a range of1-30 ng/g fresh weight (FW). In some embodiments, the mixotrophicChlorella can comprise gibberellins in a range of 0.1-1 ng/g freshweight (FW).

TABLE 3 Range Metabolite (ng/g DW) cis-Abscisic acid  0.1-13 Abscisicacid glucose 0.1-5 ester Phaseic acid 0.1-9 Neo-Phaseic acid 0.1-5trans-Abscisic acid 0.1-8 (trans) Zeatin 0.1-5 (cis) Zeatin  0.1-16(trans) Zeatin   4-20 riboside (cis) Zeatin riboside   30-250Dihydrozeatin 0.1-2 riboside Isopentenyladenine 0.1-8Isopentenyladenosine   1-15 Indole-3-acetic acid   400-815N-(Indole-3-yl- 0.1-5 acetyl)-alanine gibberellin 3 0.1-5 gibberellin 340.1-5 gibberellin 44 0.1-5

After harvest of the mixotrophic Chlorella composition from theculturing vessel, the mixotrophic Chlorella based composition istypically incorporated in a liquid composition for application toplants. Generally, the liquid composition is stabilized by heating andcooling in a pasteurization process, adjustment of pH, and the additionof an inhibitor of yeast and mold growth.

In some embodiments, the mixotrophic Chlorella can be previously frozenand thawed before inclusion in the liquid composition. In someembodiments, the mixotrophic Chlorella has not been subjected to aprevious freezing or thawing process. In some embodiments, themixotrophic Chlorella whole cells have not been subjected to a dryingprocess. In some embodiments, the cell walls of the mixotrophicChlorella of the composition have not been lysed or disrupted, and themixotrophic Chlorella cells have not been subjected to an extractionprocess or process that pulverizes the cells. The mixotrophic Chlorellawhole cells typically are not subjected to a purification process forisolating the mixotrophic Chlorella whole cells from the accompanyingconstituents of the culturing process (e.g., trace nutrients, residualorganic carbon, bacteria, cell debris, cell excretions), and thus thewhole output from the mixotrophic Chlorella culturing process comprisingwhole Chlorella cells, culture medium, cell excretions, cell debris,bacteria, residual organic carbon, and trace nutrients, is used in theliquid composition for application to plants. In some embodiments, themixotrophic Chlorella whole cells and the accompanying constituents ofthe culturing process are concentrated in the composition. In someembodiments, the mixotrophic Chlorella whole cells and the accompanyingconstituents of the culturing process are diluted in the composition toa low concentration. The mixotrophic Chlorella whole cells of thecomposition typically are not fossilized. In some embodiments, themixotrophic Chlorella whole cells typically are not maintained in aviable state in the composition for continued growth after the method ofusing the composition in a soil or foliar application. In someembodiments, the mixotrophic Chlorella base composition can bebiologically inactive after the composition is prepared. In someembodiments, the mixotrophic Chlorella base composition can besubstantially biologically inactive after the composition is prepared.In some embodiments, the mixotrophic Chlorella base composition canincrease in biological activity after the prepared composition isexposed to air.

In one non-limiting example of preparing the liquid composition with themixotrophic Chlorella based composition for application to plants, themixotrophic Chlorella based composition harvested from the culturingsystem is first held in a harvest tank before centrifuging the culture.Once the mixotrophic Chlorella culture is centrifuged, the centrifugedischarges the fraction rich in mixotrophic Chlorella whole cell solids,but also containing the accompanying constituents from the culturemedium, into a container at a temperature of about 30° C. Themixotrophic Chlorella based composition can continue (i.e., fresh) inthe process of preparing the liquid composition or be stored in afreezer and thawed at a later time (i.e., stored) for processing intothe liquid composition. When the mixotrophic Chlorella based compositionis stored in a freezer, the storage temperature is about −10° C. and ittakes about 1-2 days for the composition to freeze. Once removed fromthe freezer, the stored mixotrophic Chlorella based composition isplaced outside to thaw for about 7 days. The fresh or stored mixotrophicChlorella based composition is then placed in a tank and heated to atemperature of about 60° C. for about 2 hours to begin thepasteurization process. The mixotrophic Chlorella based composition isthen diluted to a whole cells solids concentration of about 10-11% byweight and cooled to about 40° C. to complete the pasteurizationprocess. The pH of the mixotrophic Chlorella based composition is thenadjusted to a pH of about 4 by mixing in an effective amount ofphosphoric acid for stabilization purposes. About 0.3% potassium sorbateis then mixed with the mixotrophic Chlorella based composition forstabilization purposes. The resulting liquid composition is thentransferred to containers of a desired size stored at 3-5° C. untilshipped.

While a similar process for preparing a liquid composition with themixotrophically cultured Chlorella for application to plants can beperformed with an additional step of drying the microalgae aftercentrifugation, the inventors surprisingly found that liquidcompositions containing microalgae that was not dried produced bettereffects when applied to plants. Such effects found by the inventors tobe increased when the mixotrophic Chlorella was not dried comprised:accelerated germination, chlorophyll content, and shoot weight. Theinventors also found that the subjecting the mixotrophic Chlorella to adrum drying process lowered the detectable levels of phytohormones inthe microalgae biomass.

While separate active ingredients are not added to or supplemented inthe mixotrophic Chlorella based composition, the liquid compositioncomprising the mixotrophic Chlorella whole cells and accompanyingconstituents from the culturing medium and process (e.g., tracenutrients, residual organic carbon, bacteria, cell debris, cellexcretions) can be stabilized by heating and cooling in a pasteurizationprocess. As shown in the Examples, the inventors found that the activeingredients of the mixotrophic Chlorella based composition maintainedeffectiveness in improving plant germination, emergence, maturation, andgrowth when applied to plants after being subjected to the heating andcooling of a pasteurization process and also observed variousperformance enhancements arising from pasteurization as compared to anon-pasteurized version of the formulation.

While the mixotrophic Chlorella cells are intact and viable (i.e.,physically fit to live, capable of further growth or cell division)after being harvested from the culture, the Chlorella cells resultingfrom the pasteurization process were confirmed to have intact cell wallsbut were not viable. Mixotrophic Chlorella cells resulting from thepasteurization process were observed under a microscope to determine thecondition of the cell walls after the being subjected to the heating andcooling of the process, and was visually confirmed that the Chlorellacell walls were intact and not broken open. For further investigation ofthe condition of the cell, a culture of live mixotrophic Chlorella cellsand the mixotrophic Chlorella cells resulting from the pasteurizationprocess were subjected to propidium iodide, an exclusion fluorescent dyethat labels DNA if the cell membrane is compromised, and visuallycompared under a microscope. The propidium iodide comparison showed thatthe Chlorella cells resulting from the pasteurization process containeda high amount of dyed DNA, resulting in the conclusion that themixotrophic Chlorella cell walls were intact but the cell membranes werecompromised (FIG. 2). Thus, the permeability of the pasteurizedChlorella cells differs from the permeability of a Chlorella cell withboth an intact cell wall and cell membrane.

Additionally, a culture of live mixotrophic Chlorella cells and themixotrophic Chlorella cells resulting from the pasteurization processwere subjected to DAPI (4′,6-diamidino-2-phyenylindole)-DNA bindingfluorescent dye and visually compared under a microscope. The DAPI-DNAbinding dye comparison showed that the Chlorella cells resulting fromthe pasteurization process contained a greatly diminished amount ofviable DNA in the cells, indicating that the mixotrophic Chlorella cellsare not viable after pasteurization (FIG. 1). The two DNA dyingcomparisons demonstrate that the pasteurization process transformed thestructure and function of the Chlorella cells from the natural state bychanging: the cells from viable to non-viable, the condition of the cellmembrane, and the permeability of the cells.

In some embodiments, the microalgae based composition can be heated to atemperature in the range of 50-90° C. In some embodiments, themicroalgae based composition can be heated to a temperature in the rangeof 55-65° C. In some embodiments, the microalgae based composition canbe heated to a temperature in the range of 58-62° C. In someembodiments, the microalgae based composition can be heated to atemperature in the range of 50-60° C. In some embodiments, themicroalgae based composition can be heated to a temperature in the rangeof 60-70° C. In some embodiments, the microalgae based composition canbe heated to a temperature in the range of 70-80° C. In someembodiments, the microalgae based composition can be heated to atemperature in the range of 80-90° C.

In some embodiments, the microalgae based composition can be heated fora time period in the range of 90-150 minutes. In some embodiments, themicroalgae based composition can be heated for a time period in therange of 110-130 minutes. In some embodiments, the microalgae basedcomposition can be heated for a time period in the range of 90-100minutes. In some embodiments, the microalgae based composition can beheated for a time period in the range of 100-110 minutes. In someembodiments, the microalgae based composition can be heated for a timeperiod in the range of 110-120 minutes. In some embodiments, themicroalgae based composition can be heated for a time period in therange of 120-130 minutes. In some embodiments, the microalgae basedcomposition can be heated for a time period in the range of 130-140minutes. In some embodiments, the microalgae based composition can beheated for a time period in the range of 140-150 minutes.

In some embodiments, the microalgae based composition can be heated fora time period in the range of 15-360 minutes. In some embodiments, themicroalgae based composition can be heated for a time period in therange of 15-30 minutes. In some embodiments, the microalgae basedcomposition can be heated for a time period in the range of 30-60minutes. In some embodiments, the microalgae based composition can beheated for a time period in the range of 60-120 minutes. In someembodiments, the microalgae based composition can be heated for a timeperiod in the range of 120-180 minutes. In some embodiments, themicroalgae based composition can be heated for a time period in therange of 180-360 minutes.

In some embodiments, the microalgae based composition can be cooled to atemperature in the range of 35-45° C. In some embodiments, themicroalgae based composition can be cooled to a temperature in the rangeof 36-44° C. In some embodiments, the microalgae based composition canbe cooled to a temperature in the range of 37-43° C. In someembodiments, the microalgae based composition can be cooled to atemperature in the range of 38-42° C. In some embodiments, themicroalgae based composition can be cooled to a temperature in the rangeof 39-41° C. In some embodiments, the microalgae based composition canbe cooled to a temperature suitable for further processing or handling.

In some embodiments, a method of preparing a low concentrationmixotrophic Chlorella based liquid composition for application to plantscan comprise: culturing Chlorella in an liquid culture medium andmixotrophic conditions comprising utilization of an organic carbonsource and photosynthetically active radiation as energy sources in aculturing vessel; harvesting the mixotrophic Chlorella culture from theculturing vessel; and mixing the mixotrophic Chlorella culture with anacid and a yeast and mold inhibitor to form a composition with aconcentration of an effective amount of the mixotrophic Chlorella basedcomposition for application to a plant for enhanced characteristics,wherein the whole mixotrophic Chlorella cells have not been subjected toa drying process.

In some embodiments, a method of preparing a mixotrophic Chlorella basedliquid composition for application to plants can comprise: heating acomposition comprising whole microalgae cells in an liquid medium at atemperature in the range of 50-70° C.; adjusting concentration of thewhole cells in the heated composition to a concentration in the range of5-30% whole microalgae cells by weight; cooling the composition to atemperature in the range of 35-45° C.; adjusting the pH of thecomposition to a pH in the range of 3-5; and contacting the compositionwith a yeast and mold inhibitor.

In some embodiments, the composition can comprise 5-30% solids by weightof whole mixotrophic Chlorella cells. In some embodiments, thecomposition can comprise 5-20% solids by weight of whole mixotrophicChlorella cells. In some embodiments, the composition can comprise 5-15%solids by weight of whole mixotrophic Chlorella cells. In someembodiments, the composition can comprise 5-10% solids by weight ofwhole mixotrophic Chlorella cells. In some embodiments, the compositioncan comprise 10-20% solids by weight of whole mixotrophic Chlorellacells. In some embodiments, the composition can comprise 10-20% solidsby weight of whole mixotrophic Chlorella cells. In some embodiments, thecomposition can comprise 20-30% solids by weight of whole mixotrophicChlorella cells. In some embodiments, further dilution of the wholemixotrophic Chlorella cells percent solids by weight can be occur beforeapplication for low concentration applications of the composition.

The composition can be diluted to a lower concentration for an effectiveamount in a soil or foliar application by mixing a volume of thecomposition in a volume of water. The percent solids of mixotrophicChlorella whole cells resulting in the diluted composition can becalculated by multiplying the original percent solids of mixotrophicChlorella whole cells in the composition by the ratio of the volume ofthe composition to the volume of water.

In some embodiments, the composition can comprise less than 1% solids byweight of whole mixotrophic Chlorella cells. In some embodiments, thecomposition can comprise less than 0.9% solids by weight of wholemixotrophic Chlorella cells. In some embodiments, the composition cancomprise less than 0.8% solids by weight of whole mixotrophic Chlorellacells. In some embodiments, the composition can comprise less than 0.7%solids by weight of whole mixotrophic Chlorella cells. In someembodiments, the composition can comprise less than 0.6% solids byweight of whole mixotrophic Chlorella cells. In some embodiments, thecomposition can comprise less than 0.5% solids by weight of wholemixotrophic Chlorella cells. In some embodiments, the composition cancomprise less than 0.4% solids by weight of whole mixotrophic Chlorellacells. In some embodiments, the composition can comprise less than 0.3%solids by weight of whole mixotrophic Chlorella cells. In someembodiments, the composition can comprise less than 0.2% solids byweight of whole mixotrophic Chlorella cells. In some embodiments, thecomposition can comprise less than 0.1% solids by weight of wholemixotrophic Chlorella cells. In some embodiments, the effective amountin an application of the liquid composition for enhanced germination,emergence, or maturation of a plant can comprise a concentration ofsolids of mixotrophic Chlorella whole cells in the range of0.002642-0.079252% (e.g., about 0.003% to about 0.080%), equivalent to adiluted concentration of 2-10 mL/gallon of a solution with an originalpercent solids of mixotrophic Chlorella whole cells in the range of5-30%.

In some embodiments, the effective amount in an application of theliquid composition can comprise a concentration in the range of 1-50mL/gallon, resulting in a reduction of the percent solids of mixotrophicChlorella whole cells from 5-30% to 0.001321-0.396258% (e.g., about0.001% to about 0.400%). In some embodiments, the effective amount in anapplication of the liquid composition can comprise a concentration inthe range of 1-10 mL/gallon, resulting in a reduction of the percentsolids of mixotrophic Chlorella whole cells from 5-30% to0.001321-0.079252% (e.g., about 0.001% to about 0.080%). In someembodiments, the effective amount in an application of the liquidcomposition can comprise a concentration in the range of 2-7 mL/gallon,resulting in a reduction of the percent solids of mixotrophic Chlorellawhole cells from 5-30% to 0.002642-0.055476% (e.g., about 0.003% toabout 0.055%). In some embodiments, the effective amount in anapplication of the liquid composition can comprise a concentration inthe range of 10-20 mL/gallon, resulting in a reduction of the percentsolids of mixotrophic Chlorella whole cells from 5-30% to0.013201-0.158503% (e.g., about 0.013% to about 0.160%). In someembodiments, the effective amount in an application of the liquidcomposition can comprise a concentration in the range of 20-30mL/gallon, resulting in a reduction of the percent solids of mixotrophicChlorella whole cells from 5-30% to 0.026417-0.237755% (e.g., about0.025% to about 0.250%). In some embodiments, the effective amount in anapplication of the liquid composition can comprise a concentration inthe range of 30-45 mL/gallon, resulting in a reduction of the percentsolids of mixotrophic Chlorella whole cells from 5-30% to0.039626-0.356631% (e.g., about 0.040% to about 0.360%). In someembodiments, the effective amount in an application of the liquidcomposition can comprise a concentration in the range of 30-40mL/gallon, resulting in a reduction of the percent solids of mixotrophicChlorella whole cells from 5-30% to 0.039626-0.317007% (e.g., about0.040% to about 0.320%). In some embodiments, the effective amount in anapplication of the liquid composition can comprise a concentration inthe range of 40-50 mL/gallon, resulting in a reduction of the percentsolids of mixotrophic Chlorella whole cells from 5-30% to0.052834-0.396258% (e.g., about 0.055% to about 0.400%).

In some embodiments, the liquid composition can comprise lowconcentrations of bacteria contributing to the solids percentage of thecomposition in addition to the whole mixotrophic Chlorella cells.Examples of bacteria found in non-axenic mixotrophic conditions can befound in WO2014/074769A2 (Ganuza, et al.), hereby incorporated byreference. A live bacteria count can be determined using methods knownin the art such as plate counts, plates counts using Petrifilm availablefrom 3M (St. Paul, Minn.), spectrophotometric (turbidimetric)measurements, visual comparison of turbidity with a known standard,direct cell counts under a microscope, cell mass determination, andmeasurement of cellular activity. Live bacteria counts in a non-axenicmixotrophic microalgae culture can range from 10⁴ to 10⁹ CFU/mL, and candepend on contamination control measures taken during the culturing ofthe microalgae. The level of bacteria in the composition can bedetermined by an aerobic plate count which quantifies aerobic colonyforming units (CFU) in a designated volume. In some embodiments, thecomposition comprises an aerobic plate count of 40,000-400,000 CFU/mL.In some embodiments, the composition comprises an aerobic plate count of40,000-100,000 CFU/mL. In some embodiments, the composition comprises anaerobic plate count of 100,000-200,000 CFU/mL. In some embodiments, thecomposition comprises an aerobic plate count of 200,000-300,000 CFU/mL.In some embodiments, the composition comprises an aerobic plate count of300,000-400,000 CFU/mL.

Using QPCR (quantitative polymerase chain reaction) to analyze thebacteria population in a mixotrophic Chlorella culture beforepasteurization and after pasteurization, it was observed that theprofile of bacteria in the culture changes after pasteurization.Particularly, the post-pasteurization profile of bacteria includes ahigher proportion of spore forming bacteria and includes, but is notlimited to, Paenibacillus sp., Bacillus sp., Lactobacillus sp., andBrevibacillus sp as the dominant types of bacteria. Comparing theaerobic plate counts of a mixotrophic Chlorella culture beforepasteurization and after pasteurization, it was also observed that thetotal number of bacteria in the culture is lower after pasteurization.Combinations of temperature and time for the pasteurization process forthe times of 15, 30, 60, 120, 180, and 360 minutes, and 50, 60, 70, 80,and 90° C. were tested with a culture of mixotrophic Chlorella, and theresulting aerobic plate counts ranged from 7.58×10⁶ CFU to as low as1.74×10³ CFU. Storage temperature was also shown to vary the profile ofbacteria of a pasteurized culture of mixotrophic Chlorella, with samplesstored at temperatures of 2-4° C., 25° C., and 40° C. varying in theaerobic plate count numbers and type of dominant bacteria species overtime.

In some embodiments, the pH of the microalgae based composition can beadjusted downward to a pH in the range of 3-5. In some embodiments, thepH of the microalgae based composition can be adjusted upward to a pH inthe range of 3-5. In some embodiments, the pH of the microalgae basedcomposition can be adjusted to a pH in the range of 3.5-4.5. In someembodiments, the pH of the microalgae based composition can be adjustedto a pH in the range of 3-3.5. In some embodiments, the pH of themicroalgae based composition can be adjusted to a pH in the range of3.5-4. In some embodiments, the pH of the microalgae based compositioncan be adjusted to a pH in the range of 4-4.5. In some embodiments, thepH of the microalgae based composition can be adjusted to a pH in therange of 4.5-5.

In some embodiments, stabilizing means that are not active regarding theimprovement of plant germination, emergence, and maturation, but insteadaid in stabilizing the microalgae based composition can be added toprevent the proliferation of unwanted microorganisms (e.g., yeast, mold)and prolong shelf life. Such inactive but stabilizing means can comprisean acid, and a yeast and mold inhibitor. In some embodiments, thestabilizing means are suitable for plants and do not inhibit the growthor health of the plant. In the alternative, the stabilizing means cancontribute to nutritional properties of the liquid composition, such asbut not limited to, the levels of nitrogen, phosphorus, or potassium.

In some embodiments, the step of adjusting the pH of the compositioncomprises contacting the composition with stabilizing means comprisingan acid. In some embodiments, such an acid can comprise phosphoric acid(H₃PO₄). In some embodiments, the amount of acid needed to adjust the pHcan comprise different amounts of acid depending on the starting pH ofthe microalgae composition, which can vary based on culturing conditionsof the microalgae, residual concentrations of organic carbon or othernutrients, and previous processing of the composition. In someembodiments, the microalgae based composition can comprise less than0.3% phosphoric acid. In some embodiments, the microalgae basedcomposition can comprise 0.01-0.3% phosphoric acid. In some embodiments,the microalgae based composition can comprise 0.05-0.25% phosphoricacid. In some embodiments, the microalgae based composition can comprise0.01-0.1% phosphoric acid. In some embodiments, the microalgae basedcomposition can comprise 0.1-0.2% phosphoric acid. In some embodiments,the microalgae based composition can comprise 0.2-0.3% phosphoric acid.

In some embodiments, the yeast and mold inhibitor can comprise potassiumsorbate (C₆H₇KO₂). In some embodiments, the composition can compriseless than 0.5% potassium sorbate. In some embodiments, the compositioncan comprise 0.01-0.5% potassium sorbate. In some embodiments, thecomposition can comprise 0.05-0.4% potassium sorbate. In someembodiments, the composition can comprise 0.01-0.1% potassium sorbate.In some embodiments, the composition can comprise 0.1-0.2% potassiumsorbate. In some embodiments, the composition can comprise 0.2-0.3%potassium sorbate. In some embodiments, the composition can comprise0.3-0.4% potassium sorbate. In some embodiments, the composition cancomprise 0.4-0.5% potassium sorbate. In other embodiments, thestabilizing function of potassium sorbate and/or phosphoric acid can beachieved with use of comparable additives with similar function such as,for example, ascorbic acid, sodium benzoate, or the like, inquantities/concentrations similar to those listed herein for potassiumsorbate and phosphoric acid.

The microalgae based composition is a liquid and is substantiallycomprised of water. In some embodiments, the composition can comprise70-95% water. In some embodiments, the composition can comprise 85-95%water. In some embodiments, the composition can comprise 70-75% water.In some embodiments, the composition can comprise 75-80% water. In someembodiments, the composition can comprise 80-85% water. In someembodiments, the composition can comprise 85-90% water. In someembodiments, the composition can comprise 90-95% water. The liquidnature and high water content of the microalgae based compositionfacilitates administration of the composition in a variety of manners,such as but not limited to: flowing through an irrigation system,flowing through an above ground drip irrigation system, flowing througha buried drip irrigation system, flowing through a central pivotirrigation system, sprayers, sprinklers, water cans, and the like.

The microalgae based composition can be used immediately afterformulation, or can be stored in containers for later use. In someembodiments, the microalgae based composition can be stored out ofdirect sunlight. In some embodiments, the microalgae based compositioncan be refrigerated. In some embodiments, the microalgae basedcomposition can be stored at 1-10° C. In some embodiments, themicroalgae based composition can be stored at 1-3° C. In someembodiments, the microalgae based composition can be stored at 3-5° C.In some embodiments, the microalgae based composition can be stored at5-8° C. In some embodiments, the composition can be stored at 8-10° C.

In some embodiments, not drying the mixotrophic Chlorella basedcomposition during preparation can increase seed emergence by 40-4,000%,or more, for soil applications. In some embodiments, not drying themixotrophic Chlorella based composition during preparation can increasechlorophyll content by 10-30% for foliar applications. In someembodiments, not drying the mixotrophic Chlorella based compositionduring preparation can increase whole plant weight emergence by 10-20%for foliar applications. In some embodiments, not drying the mixotrophicChlorella based composition during preparation can increase shoot weightby 20-30% for foliar applications.

Administration of the mixotrophic Chlorella based liquid compositiontreatment to a seed or plant can be in an amount effective to produce anenhanced characteristic in the plant compared to a substantiallyidentical population of untreated plant. Such enhanced characteristicscan comprise accelerated seed germination, accelerated seedlingemergence, improved seedling emergence, improved leaf formation,accelerated leaf formation, improved plant maturation, accelerated plantmaturation, increased plant yield, increased plant growth, increasedplant quality, increased plant health, increased fruit yield, increasedfruit growth, and increased fruit quality. Non-limiting examples of suchenhanced characteristics can comprise accelerated achievement of thehypocotyl stage, accelerated protrusion of a stem from the soil,accelerated achievement of the cotyledon stage, accelerated leafformation, increased marketable plant weight, increased marketable plantyield, increased marketable fruit weight, increased production plantweight, increased production fruit weight, increased utilization(indicator of efficiency in the agricultural process based on ratio ofmarketable fruit to unmarketable fruit), increased chlorophyll content(indicator of plant health), increased plant weight (indicator of planthealth), increased root weight (indicator of plant health), andincreased shoot weight (indicator of plant health). Such enhancedcharacteristics can occur individually in a plant, or in combinations ofmultiple enhanced characteristics.

Surprisingly, the inventors found that administration of the describedmixotrophic Chlorella based composition in low concentrationapplications was effective in producing enhanced characteristics inplants. In some embodiments, the mixotrophic Chlorella based liquidcomposition treatment is administered before the seed is planted. Insome embodiments, the mixotrophic Chlorella based liquid compositiontreatment is administered at the time the seed is planted. In someembodiments, the mixotrophic Chlorella based liquid compositiontreatment is administered after the seed is planted, including forexample, at various post-emergence growth and maturation stages of theplant.

In some embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the number of plants emerged by 20-160%,or more, compared to a substantially identical population of untreatedseeds or plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the number of plantsemerged by at least 20% compared to a substantially identical populationof untreated seeds or plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase the numberof plants emerged by at least 40% compared to a substantially identicalpopulation of untreated seeds or plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number of plants emerged by at least 60% compared to asubstantially identical population of untreated seeds or plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number of plants emerged by at least 80%compared to a substantially identical population of untreated seeds orplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the number of plants emerged by atleast 100% compared to a substantially identical population of untreatedseeds or plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the number of plantsemerged by at least 120% compared to a substantially identicalpopulation of untreated seeds or plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number of plants emerged by at least 140% compared to asubstantially identical population of untreated seeds or plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number of plants emerged by at least 150%compared to a substantially identical population of untreated seeds orplants.

In some embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the number of plants emerged by25-2000%, or more, compared to a substantially identical population ofuntreated seeds of plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase the numberof plants emerged by at least 25% compared to a substantially identicalpopulation of untreated seeds of plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number of plants emerged by at least 30% compared to asubstantially identical population of untreated seeds of plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number of plants emerged by at least 40%compared to a substantially identical population of untreated seeds ofplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the number of plants emerged by atleast 50% compared to a substantially identical population of untreatedseeds of plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the number of plantsemerged by at least 60% compared to a substantially identical populationof untreated seeds of plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase the numberof plants emerged by at least 70% compared to a substantially identicalpopulation of untreated seeds of plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number of plants emerged by at least 80% compared to asubstantially identical population of untreated seeds of plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number of plants emerged by at least 90%compared to a substantially identical population of untreated seeds ofplants.

In some embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the number of plants emerged by at least100% compared to a substantially identical population of untreated seedsof plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the number of plantsemerged by at least 200% compared to a substantially identicalpopulation of untreated seeds of plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number of plants emerged by at least 300% compared to asubstantially identical population of untreated seeds of plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number of plants emerged by at least 400%compared to a substantially identical population of untreated seeds ofplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the number of plants emerged by atleast 500% compared to a substantially identical population of untreatedseeds of plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the number of plantsemerged by at least 600% compared to a substantially identicalpopulation of untreated seeds of plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number of plants emerged by at least 700% compared to asubstantially identical population of untreated seeds of plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number of plants emerged by at least 800%compared to a substantially identical population of untreated seeds ofplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the number of plants emerged by atleast 900% compared to a substantially identical population of untreatedseeds of plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the number of plantsemerged by at least 1,000% compared to a substantially identicalpopulation of untreated seeds of plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number of plants emerged by at least 1,500% compared to asubstantially identical population of untreated seeds of plants.

In some embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the number of plants demonstratingmaturation by leaf formation by 30-180%, or more, compared to asubstantially identical population of untreated seeds or plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number plants demonstrating maturation byleaf formation by at least 30% compared to a substantially identicalpopulation of untreated seeds or plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number plants demonstrating maturation by leaf formation byat least 50% compared to a substantially identical population ofuntreated seeds or plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase the numberplants demonstrating maturation by leaf formation by at least 70%compared to a substantially identical population of untreated seeds orplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the number plants demonstratingmaturation by leaf formation by at least 90% compared to a substantiallyidentical population of untreated seeds or plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number plants demonstrating maturation by leaf formation byat least 110% compared to a substantially identical population ofuntreated seeds or plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase the numberplants demonstrating maturation by leaf formation by at least 130%compared to a substantially identical population of untreated seeds orplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the number plants demonstratingmaturation by leaf formation by at least 150% compared to asubstantially identical population of untreated seeds or plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number plants demonstrating maturation byleaf formation by at least 160% compared to a substantially identicalpopulation of untreated seeds or plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number plants demonstrating maturation by leaf formation byat least 170% compared to a substantially identical population ofuntreated seeds or plants.

In some embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the number of plants demonstratingmaturation by leaf formation by 20-350%, or more, compared to asubstantially identical population of untreated seeds of plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number plants demonstrating maturation byleaf formation by at least 20% compared to a substantially identicalpopulation of untreated seeds of plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number plants demonstrating maturation by leaf formation byat least 30% compared to a substantially identical population ofuntreated seeds of plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase the numberplants demonstrating maturation by leaf formation by at least 40%compared to a substantially identical population of untreated seeds ofplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the number plants demonstratingmaturation by leaf formation by at least 50% compared to a substantiallyidentical population of untreated seeds of plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number plants demonstrating maturation by leaf formation byat least 60% compared to a substantially identical population ofuntreated seeds of plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase the numberplants demonstrating maturation by leaf formation by at least 70%compared to a substantially identical population of untreated seeds ofplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the number plants demonstratingmaturation by leaf formation by at least 80% compared to a substantiallyidentical population of untreated seeds of plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number plants demonstrating maturation by leaf formation byat least 90% compared to a substantially identical population ofuntreated seeds of plants.

In some embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the number of plants demonstratingmaturation by leaf formation by at least 100% compared to asubstantially identical population of untreated seeds of plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the number plants demonstrating maturation byleaf formation by at least 150% compared to a substantially identicalpopulation of untreated seeds of plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the number plants demonstrating maturation by leaf formation byat least 200% compared to a substantially identical population ofuntreated seeds of plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase the numberplants demonstrating maturation by leaf formation by at least 250%compared to a substantially identical population of untreated seeds ofplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the number plants demonstratingmaturation by leaf formation by at least 300% compared to asubstantially identical population of untreated seeds of plants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase utilization by 80-100% compared toa substantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase utilization by at least 80% compared to asubstantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase utilization by at least 85% compared to asubstantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase utilization by at least 90% compared to asubstantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase utilization by at least 95% compared to asubstantially identical population of untreated plants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase marketable plant weight by200-290%, or more, compared to a substantially identical population ofuntreated plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the marketable plantweight by at least 200% compared to a substantially identical populationof untreated plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase themarketable plant weight by at least 210% compared to a substantiallyidentical population of untreated plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the marketable plant weight by at least 220% compared to asubstantially identical population of untreated plants. In someembodiments administration of the mixotrophic Chlorella based liquidcomposition can increase the marketable plant weight by at least 230%compared to a substantially identical population of untreated plants. Insome embodiments administration of the mixotrophic Chlorella basedliquid composition can increase the marketable plant weight by at least240% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable plant weight by atleast 250% compared a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable plant weight by atleast 260% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable plant weight by atleast 270% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable plant weight by atleast 280% compared to a substantially identical population of untreatedplants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase marketable plant yield by150-230%, or more, compared to a substantially identical population ofuntreated plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the marketable plantyield by at least 150% compared to a substantially identical populationof untreated plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase themarketable plant yield by at least 180% compared to a substantiallyidentical population of untreated plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the marketable plant yield by at least 190% compared to asubstantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the marketable plant yield by at least 200%compared to a substantially identical population of untreated plants. Insome embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the marketable plant yield by at least210% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable plant yield by atleast 220% compared to a substantially identical population of untreatedplants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase marketable fruit weight by 10-50%,or more, compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable fruit weight by atleast 10% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable fruit weight by atleast 20% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable fruit weight by atleast 30% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable fruit weight by atleast 40% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the marketable fruit weight by atleast 45% compared to a substantially identical population of untreatedplants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase production plant weight by70-120%, or more, compared to a substantially identical population ofuntreated plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase production plant weightby at least 70% compared to a substantially identical population ofuntreated plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the production plantweight by at least 80% compared to a substantially identical populationof untreated plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase theproduction plant weight by at least 90% compared to a substantiallyidentical population of untreated plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the production plant weight by at least 100% compared to asubstantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the production plant weight by at least 110%compared to a substantially identical population of untreated plants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase production fruit weight by70-110%, or more, compared to a substantially identical population ofuntreated plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase production fruit weightby at least 70% compared to a substantially identical population ofuntreated plants. In some embodiments, administration of the mixotrophicChlorella based liquid composition can increase the production fruitweight by at least 80% compared to a substantially identical populationof untreated plants. In some embodiments, administration of themixotrophic Chlorella based liquid composition can increase theproduction fruit weight by at least 90% compared to a substantiallyidentical population of untreated plants. In some embodiments,administration of the mixotrophic Chlorella based liquid composition canincrease the production fruit weight by at least 100% compared to asubstantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the production fruit weight by at least 105%compared to a substantially identical population of untreated plants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase the chlorophyll content by 15-40%,or more, compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the chlorophyll content by atleast 15% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the chlorophyll content by atleast 20% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the chlorophyll content by atleast 25% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the chlorophyll content by atleast 30% compared to a substantially identical population of untreatedplants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase the whole plant weight by 30-60%,or more, compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the whole plant weight by at least30% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the whole plant weight by at least35% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the whole plant weight by at least40% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the whole plant weight by at least45% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the whole plant weight by at least50% compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the whole plant weight by at least55% compared to a substantially identical population of untreatedplants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase the root weight by 30-60%, ormore, compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the root weight by at least 30%compared to a substantially identical population of untreated plants. Insome embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the root weight by at least 35% comparedto a substantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the root weight by at least 40% compared to asubstantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the root weight by at least 45% compared to asubstantially identical population of untreated plants. In someembodiments, administration of the mixotrophic Chlorella based liquidcomposition can increase the root weight by at least 50% compared to asubstantially identical population of untreated plants.

In some embodiments, the administration of the mixotrophic Chlorellabased liquid composition can increase the shoot weight by 30-70%, ormore, compared to a substantially identical population of untreatedplants. In some embodiments, administration of the mixotrophic Chlorellabased liquid composition can increase the shoot weight by at least 30%compared to a substantially identical population of untreated plants. Insome embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the shoot weight by at least 35%compared to a substantially identical population of untreated plants. Insome embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the shoot weight by at least 40%compared to a substantially identical population of untreated plants. Insome embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the shoot weight by at least 45%compared to a substantially identical population of untreated plants. Insome embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the shoot weight by at least 50%compared a substantially identical population of untreated plants. Insome embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the shoot weight by at least 55%compared to a substantially identical population of untreated plants. Insome embodiments, administration of the mixotrophic Chlorella basedliquid composition can increase the shoot weight by at least 60%compared to a substantially identical population of untreated plants.

Whether in a seed soak, capillary action, soil, or foliar applicationthe method of use comprises relatively low concentrations of themixotrophic Chlorella based liquid composition. Even at such lowconcentrations, the described composition has been shown to be effectiveat producing an enhanced characteristic in plants. The ability to uselow concentrations allows for a reduced impact on the environment thatcan result from over application and an increased efficiency in themethod of use of the liquid composition by requiring a small amount ofmaterial to produce the desired effect. In some embodiments, the use ofthe mixotrophic Chlorella based liquid composition with a low volumeirrigation system in soil applications allows the low concentration ofthe liquid composition to remain effective and not be diluted to a pointwhere the composition is no longer in at a concentration capable ofproducing the desired effect on the plants while also increasing thegrower's water use efficiency. The ability to use low concentrations ofmixotrophic Chlorella whole cells and lack of purification processes toisolate the cells also reduces the dewatering and processing needs ofthe microalgae which can be produced at low concentrations in theculturing stage, and thus increasing the energy efficiency in the methodof preparing the product.

In conjunction with the low concentrations of mixotrophic Chlorellawhole cells solids in the liquid composition necessary to be effectivefor enhancing the described characteristics of plants, the liquidcomposition can does not have be to administered continuously or at ahigh frequency (e.g., multiple times per day, daily). The ability of themixotrophic Chlorella based liquid composition to be effective at lowconcentrations and a low frequency of application was an unexpectedresult, due to the traditional thinking that as the concentration ofactive ingredients decreases the frequency of application shouldincrease to provide adequate amounts of the active ingredients.Effectiveness at low concentration and application frequency increasesthe material usage efficiency of the method of using the liquidcomposition while also increasing the yield efficiency of theagricultural process. The use a composition of mixotrophic Chlorellawhole cells that does not require processing to dry, extract, lyse, orotherwise disrupt the cell wall also increases energy efficiency in themethod of preparing the product and allows the product to be produced ina quicker time frame.

Seed Soak Application

In one non-limiting embodiment, the administration of the mixotrophicChlorella based liquid composition treatment can comprise soaking theseed in an effective amount of the liquid composition before plantingthe seed. In some embodiments, the administration of the mixotrophicChlorella based liquid composition further comprises removing the seedfrom the liquid composition after soaking, and drying the seed beforeplanting. In some embodiments, the seed can be soaked in the mixotrophicChlorella based liquid composition for a time period in the range of90-150 minutes. In some embodiments, the seed can be soaked in themixotrophic Chlorella based liquid composition for a time period in therange of 110-130 minutes. In some embodiments, the seed can be soaked inthe mixotrophic Chlorella based liquid composition for a time period inthe range of 90-100 minutes. In some embodiments, the seed can be soakedin the mixotrophic Chlorella based liquid composition for a time periodin the range of 100-110 minutes. In some embodiments, the seed can besoaked in the mixotrophic Chlorella based liquid composition for a timeperiod in the range of 110-120 minutes. In some embodiments, the seedcan be soaked in the mixotrophic Chlorella based liquid composition fora time period in the range of 120-130 minutes. In some embodiments, theseed can be soaked in the mixotrophic Chlorella based liquid compositionfor a time period in the range of 130-140 minutes. In some embodiments,the seed can be soaked in the mixotrophic Chlorella based liquidcomposition for a time period in the range of 140-150 minutes.

The composition can be diluted to a lower concentration for an effectiveamount in a seed soak application by mixing a volume of the mixotrophicChlorella based composition in a volume of water. The percent solids ofmixotrophic Chlorella whole cells resulting in the diluted compositioncan be calculated by multiplying the original percent solids in thecomposition by the ratio of the volume of the composition to the volumeof water. In some embodiments, the effective amount in a seed soakapplication of the mixotrophic Chlorella based liquid composition cancomprise a concentration in the range of 6-10 mL/gallon, resulting in areduction of the percent solids of mixotrophic Chlorella whole cellsfrom 5-30% to 0.007925-0.079252% (e.g., about 0.008% to about 0.080%).In some embodiments, the effective amount in a seed soak application ofthe mixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 7-9 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.009245-0.071327% (e.g., about 0.009% to about 0.070%). In someembodiments, the effective amount in a seed soak application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 6-7 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.007925-0.05547% (e.g., about 0.008% to about 0.055%). In someembodiments, the effective amount in a seed soak application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 7-8 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.009246-0.063401% (e.g., about 0.009% to about 0.065%). In someembodiments, the effective amount in a seed soak application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 8-9 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.010567-0.071327% (e.g., about 0.010% to about 0.070%). In someembodiments, the effective amount in a seed soak application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 9-10 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.011888-0.079252% (e.g., about 0.012% to about 0.080%).

Soil Application

In another non-limiting embodiment, the administration of themixotrophic Chlorella based liquid composition treatment can comprisecontacting the soil in the immediate vicinity of the planted seed orplant with an effective amount of the liquid composition. In someembodiments, the mixotrophic Chlorella based liquid composition can besupplied to the soil by injection into a low volume irrigation system,such as but not limited to a drip irrigation system supplying waterbeneath the soil through perforated conduits or at the soil level byfluid conduits hanging above the ground or protruding from the ground.In some embodiments, the mixotrophic Chlorella based liquid compositioncan be supplied to the soil by a soil drench method wherein the liquidcomposition is poured on the soil.

The mixotrophic Chlorella based composition can be diluted to a lowerconcentration for an effective amount in a soil application by mixing avolume of the composition in a volume of water. The percent solids ofmixotrophic Chlorella whole cells resulting in the diluted compositioncan be calculated by multiplying the original percent solids in thecomposition by the ratio of the volume of the composition to the volumeof water. In some embodiments, the effective amount in a soilapplication of the mixotrophic Chlorella based liquid composition cancomprise a concentration in the range of 3.5-10 mL/gallon, resulting ina reduction of the percent solids of mixotrophic Chlorella whole cellsfrom 5-30% to 0.004623-0.079252% (e.g., about 0.004% to about 0.080%).In some embodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 3.5-4 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.004623-0.031701% (e.g., about 0.004% to about 0.032%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 4-5 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.005283-0.039626% (e.g., about 0.005% to about 0.040%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 5-6 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.006604-0.047551% (e.g., about 0.006% to about 0.050%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 6-7 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.0.007925-0.055476% (e.g., about 0.008% to about 0.055%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 7-8 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.009246-0.063401% (e.g., about 0.009% to about 0.065%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 8-9 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.010567-0.071327% (e.g., about 0.010% to about 0.075%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 9-10 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.011888-0.079252% (e.g., about 0.012% to about 0.080%).

In some embodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 1-50 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.001321-0.396258% (e.g., about 0.001% to about 0.400%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 1-10 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.001321-0.079252% (e.g., about 0.001% to about 0.080%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 2-7 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.002642-0.055476% (e.g., about 0.003% to about 0.055%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 10-20 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.013201-0.158503% (e.g., about 0.013% to about 0.160%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 20-30 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.026417-0.237755% (e.g., about 0.025% to about 0.250%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 30-45 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.039626-0.356631% (e.g., about 0.040% to about 0.360%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 30-40 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.039626-0.317007% (e.g., about 0.040% to about 0.320%). In someembodiments, the effective amount in a soil application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 40-50 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.052834-0.396258% (e.g., about 0.055% to about 0.400%).

The rate of application of the mixotrophic Chlorella based compositionat the desired concentration can be expressed as a volume per area. Insome embodiments, the rate of application of the mixotrophic Chlorellabased liquid composition in a soil application can comprise a rate inthe range of 50-150 gallons/acre. In some embodiments, the rate ofapplication of the mixotrophic Chlorella based liquid composition in asoil application can comprise a rate in the range of 75-125gallons/acre. In some embodiments, the rate of application of themixotrophic Chlorella based liquid composition in a soil application cancomprise a rate in the range of 50-75 gallons/acre. In some embodiments,the rate of application of the mixotrophic Chlorella based liquidcomposition in a soil application can comprise a rate in the range of75-100 gallons/acre. In some embodiments, the rate of application of themixotrophic Chlorella based liquid composition in a soil application cancomprise a rate in the range of 100-125 gallons/acre. In someembodiments, the rate of application of the mixotrophic Chlorella basedliquid composition in a soil application can comprise a rate in therange of 125-150 gallons/acre.

The frequency of the application of the mixotrophic Chlorella basedcomposition can be expressed as the number of applications per period oftime (e.g., two applications per month), or by the period of timebetween applications (e.g., one application every 21 days). In someembodiments, the plant can be contacted by the mixotrophic Chlorellabased liquid composition in a soil application every 3-28 days. In someembodiments, the plant can be contacted by the liquid composition in asoil application every 4-10 days. In some embodiments, the plant can becontacted by the mixotrophic Chlorella based liquid composition in asoil application every 18-24 days. In some embodiments, the plant can becontacted by the liquid composition in a soil application every 3-7days. In some embodiments, the plant can be contacted by the mixotrophicChlorella based liquid composition in a soil application every 7-14days. In some embodiments, the plant can be contacted by the mixotrophicChlorella based liquid composition in a soil application every 14-21days. In some embodiments, the plant can be contacted by the mixotrophicChlorella based liquid composition in a soil application every 21-28days.

Soil application(s) of the mixotrophic Chlorella based compositiongenerally begin after the plant has become established, but can beginbefore establishment, at defined time period after planting, or at adefined time period after emergence form the soil in some embodiments.In some embodiments, the plant can be first contacted by the mixotrophicChlorella based liquid composition in a soil application 5-14 days afterthe plant emerges from the soil. In some embodiments, the plant can befirst contacted by the mixotrophic Chlorella based liquid composition ina soil application 5-7 days after the plant emerges from the soil. Insome embodiments, the plant can be first contacted by the mixotrophicChlorella based liquid composition in a soil application 7-10 days afterthe plant emerges from the soil. In some embodiments, the plant can befirst contacted by the mixotrophic Chlorella based liquid composition ina soil application 10-12 days after the plant emerges from the soil. Insome embodiments, the plant can be first contacted by the mixotrophicChlorella based liquid composition in a soil application 12-14 daysafter the plant emerges from the soil.

Capillary Action Application

In another non-limiting embodiment, the administration of themixotrophic Chlorella based liquid composition treatment can comprisefirst soaking the seed in water, removing the seed from the water,drying the seed, applying an effective amount of the liquid compositionbelow the seed planting level in the soil, and planting the seed,wherein the liquid composition supplied to the seed from below bycapillary action. In some embodiments, the seed can be soaked in waterfor a time period in the range of 90-150 minutes. In some embodiments,the seed can be soaked in water for a time period in the range of110-130 minutes. In some embodiments, the seed can be soaked in waterfor a time period in the range of 90-100 minutes. In some embodiments,the seed can be soaked in water for a time period in the range of100-110 minutes. In some embodiments, the seed can be soaked in waterfor a time period in the range of 110-120 minutes. In some embodiments,the seed can be soaked in water for a time period in the range of120-130 minutes. In some embodiments, the seed can be soaked in waterfor a time period in the range of 130-140 minutes. In some embodiments,the seed can be soaked in water for a time period in the range of140-150 minutes.

The mixotrophic Chlorella based composition can be diluted to a lowerconcentration for an effective amount in a capillary action applicationby mixing a volume of the composition in a volume of water. The percentsolids of mixotrophic Chlorella whole cells resulting in the dilutedcomposition can be calculated by multiplying the original percent solidsin the composition by the ratio of the volume of the composition to thevolume of water. In some embodiments, the effective amount in acapillary action application of the mixotrophic Chlorella based liquidcomposition can comprise a concentration in the range of 6-10 mL/gallon,resulting in a reduction of the percent solids of mixotrophic Chlorellawhole cells from 5-30% to 0.007925-0.079252% (e.g., about 0.008% toabout 0.080%). In some embodiments, the effective amount in a capillaryaction application of the mixotrophic Chlorella based liquid compositioncan comprise a concentration in the range of 7-9 mL/gallon, resulting ina reduction of the percent solids of mixotrophic Chlorella whole cellsfrom 5-30% to 0.009245-0.071327% (e.g., about 0.009% to about 0.075%).In some embodiments, the effective amount in a capillary actionapplication of the mixotrophic Chlorella based liquid composition cancomprise a concentration in the range of 6-7 mL/gallon, resulting in areduction of the percent solids of mixotrophic Chlorella whole cellsfrom 5-30% to 0.007925-0.05547% (e.g., about 0.008% to about 0.055%). Insome embodiments, the effective amount in a capillary action applicationof the mixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 7-8 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.009246-0.063401% (e.g., about 0.009% to about 0.065%). In someembodiments, the effective amount in a capillary action application ofthe mixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 8-9 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.010567-0.071327% (e.g., about 0.010% to about 0.075%). In someembodiments, the effective amount in a capillary action application ofthe mixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 9-10 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.011888-0.079252% (e.g., about 0.012% to about 0.080%).

Foliar Application

In one non-limiting embodiment, the administration of the mixotrophicChlorella based liquid composition treatment can comprise contacting thefoliage of the plant with an effective amount of the liquid composition.In some embodiments, the mixotrophic Chlorella based liquid compositioncan be sprayed on the foliage by a hand sprayer, a sprayer on anagriculture implement, a sprinkler, a broad distribution system such asa cropduster, or the like.

The mixotrophic Chlorella based composition can be diluted to a lowerconcentration for an effective amount in a foliar application by mixinga volume of the composition in a volume of water. The percent solids ofmixotrophic Chlorella whole cells resulting in the diluted compositioncan be calculated by multiplying the original percent solids in thecomposition by the ratio of the volume of the composition to the volumeof water. In some embodiments, the effective amount in a foliarapplication of the mixotrophic Chlorella based liquid composition cancomprise a concentration in the range of 2-10 mL/gallon, resulting in areduction of the percent solids of mixotrophic Chlorella whole cellsfrom 5-30% to 0.002642-0.079252% (e.g., about 0.003% to about 0.080%).In some embodiments, the effective amount in a foliar application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 2-3 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.002642-0.023775% (e.g., about 0.003% to about 0.025%). In someembodiments, the effective amount in a foliar application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 3-4 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.003963-0.031701% (e.g., about 0.004% to about 0.035%). In someembodiments, the effective amount in a foliar application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 4-5 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.005283-0.039626% (e.g., about 0.005% to about 0.040%). In someembodiments, the effective amount in a foliar application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 5-6 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.006604-0.047551% (e.g., about 0.007% to about 0.050%). In someembodiments, the effective amount in a foliar application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 6-7 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.007925-0.055476% (e.g., about 0.008% to about 0.055%). In someembodiments, the effective amount in a foliar application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 7-8 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.009246-0.063401% (e.g., about 0.009% to about 0.065%). In someembodiments, the effective amount in a foliar application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 8-9 mL/gallon, resulting in a reduction ofthe percent solids of mixotrophic Chlorella whole cells from 5-30% to0.010567-0.071327% (e.g., about 0.010% to about 0.070%). In someembodiments, the effective amount in a foliar application of themixotrophic Chlorella based liquid composition can comprise aconcentration in the range of 9-10 mL/gallon, resulting in a reductionof the percent solids of mixotrophic Chlorella whole cells from 5-30% to0.011888-0.079252% (e.g., about 0.012% to about 0.080%).

The rate of application of the mixotrophic Chlorella based compositionat the desired concentration can be expressed as a volume per area. Insome embodiments, the rate of application of the mixotrophic Chlorellabased liquid composition in a foliar application can comprise a rate inthe range of 10-50 gallons/acre. In some embodiments, the rate ofapplication of the liquid composition in a foliar application cancomprise a rate in the range of 10-15 gallons/acre. In some embodiments,the rate of application of the mixotrophic Chlorella based liquidcomposition in a foliar application can comprise a rate in the range of15-20 gallons/acre. In some embodiments, the rate of application of themixotrophic Chlorella based liquid composition in a foliar applicationcan comprise a rate in the range of 20-25 gallons/acre. In someembodiments, the rate of application of the mixotrophic Chlorella basedliquid composition in a foliar application can comprise a rate in therange of 25-30 gallons/acre. In some embodiments, the rate ofapplication of the mixotrophic Chlorella based liquid composition in afoliar application can comprise a rate in the range of 30-35gallons/acre. In some embodiments, the rate of application of themixotrophic Chlorella based liquid composition in a foliar applicationcan comprise a rate in the range of 35-40 gallons/acre. In someembodiments, the rate of application of the mixotrophic Chlorella basedliquid composition in a foliar application can comprise a rate in therange of 40-45 gallons/acre. In some embodiments, the rate ofapplication of the mixotrophic Chlorella based liquid composition in afoliar application can comprise a rate in the range of 45-50gallons/acre.

The frequency of the application of the mixotrophic Chlorella basedcomposition can be expressed as the number of applications per period oftime (e.g., two applications per month), or by the period of timebetween applications (e.g., one application every 21 days). In someembodiments, the plant can be contacted by the mixotrophic Chlorellabased liquid composition in a foliar application every 3-28 days, ormore. In some embodiments, the plant can be contacted by the mixotrophicChlorella based liquid composition in a foliar application every 4-10days. In some embodiments, the plant can be contacted by the mixotrophicChlorella based liquid composition in a foliar application every 18-24days. In some embodiments, the plant can be contacted by the mixotrophicChlorella based liquid composition in a foliar application every 3-7days. In some embodiments, the plant can be contacted by the mixotrophicChlorella based liquid composition in a foliar application every 7-14days. In some embodiments, the plant can be contacted by the mixotrophicChlorella based liquid composition in a foliar application every 14-21days. In some embodiments, the plant can be contacted by the mixotrophicChlorella based liquid composition in a foliar application every 21-28days.

Foliar application(s) of the mixotrophic Chlorella based compositiongenerally begin after the plant has become established, but can beginbefore establishment, at defined time period after planting, or at adefined time period after emergence form the soil in some embodiments.In some embodiments, the plant can be first contacted by the mixotrophicChlorella based liquid composition in a foliar application 5-14 daysafter the plant emerges from the soil. In some embodiments, the plantcan be first contacted by the mixotrophic Chlorella based liquidcomposition in a foliar application 5-7 days after the plant emergesfrom the soil. In some embodiments, the plant can be first contacted bythe mixotrophic Chlorella based liquid composition in a foliarapplication 7-10 days after the plant emerges from the soil. In someembodiments, the plant can be first contacted by the mixotrophicChlorella based liquid composition in a foliar application 10-12 daysafter the plant emerges from the soil. In some embodiments, the plantcan be first contacted by the mixotrophic Chlorella based liquidcomposition in a foliar application 12-14 days after the plant emergesfrom the soil.

Hydroponic Application

In another non-limiting embodiment, the administration of themixotrophic Chlorella based liquid composition to a seed or plant cancomprise applying the composition in combination with a nutrient mediumto seeds disposed in and plants growing in a hydroponic growth medium oran inert growth medium (e.g., coconut husks). The mixotrophic Chlorellabased liquid composition can be applied multiple times per day, perweek, or per growing season.

EXAMPLES

Embodiments of the invention are exemplified and additional embodimentsare disclosed in further detail in the following Examples, which are notin any way intended to limit the scope of any aspect of the inventiondescribed herein.

Example 1

Samples of mixotrophically cultured Chlorella whole cells were analyzedfor content. The results of the sample analysis and extrapolated rangesbased on standard deviations are shown in Table 4, with NA indicatinglevels that were too low for detection. The results of the proteinanalysis are presented on a dry weight basis, while the remainingresults are presented on a wet basis.

TABLE 4 Sample No. 1 2 3 4 Range % Protein (Leco) 34.89 35.04 29.4 24.515-45 % Lipids (AOAC) 14.6 15.3 10.75 12.9  5-20 Phosphorus (ppm) 20002300 2700 2800 1,600-3,200 Potassium (ppm) 6208 6651 7088 80085,400-9,000 Calcium (ppm) 2100 2000 1500 1200  750-2,600 Iron (ppm) 130160 140 110  80-200 Magnesium (ppm) 1500 1500 1200 970  700-1,800Manganese (ppm) 31 32 25 21 10-40 Zinc (ppm) <25 29 <25 <25 0.1-40 Arsenic (ppm) <2.5 <2.5 <2.5 <2.5 0.1-2.5 Cadmium (ppm) <0.5 1.8 <0.5<0.5 0.1-2.0 Cobalt (ppm) 2.2 1.6 1.4 1.3 0.1-5.0 Chromium (ppm) NA <1.0<1.0 <1.0 0.1-1.0 Copper (ppm) NA 180 18 14  1-300 Mercury (ppm) NA <2.0<2.0 <2.0 0.1-2.0 Molybdenum (ppm) NA <2.5 <2.5 <2.5 0.1-2.5 Sodium(ppm) 2500 5400 3300 2400 1,000-6,800 Nickel (ppm) NA <2.5 <2.5 <2.50.1-2.5 Lead (ppm) <5.0 <5.0 <5.0 <5.0 0.1-5.0 Selenium (ppm) NA <5.0<5.0 <5.0 0.1-5.0

Example 2

Samples of mixotrophically cultured Chlorella whole cells were analyzedfor amino acid content. The results of the sample analysis andextrapolated ranges are shown in Table 5.

TABLE 5 Analyte % in Biomass Range (%) Aspartic Acid 3.88 2.0-5.0Threonine 1.59 0.1-3.0 Serine 2.3 0.1-4.0 Glutamic Acid 6.01 4.0-8.0Proline 2.73 0.1-5.0 Glycine 2.45 0.1-4.0 Alanine 3.34 1.0-5.0 Cysteine0.56 0.1-2.0 Valine 1.99 0.1-4.0 Methionine 0.85 0.1-2.0 Isoleucine 1.390.1-3.0 Leucine 3.13 1.0-5.0 Tyrosine 1.50 0.1-3.0 Phenylalanine 1.770.1-4.0 Lysine 1.87 0.1-3.0 Histidine 0.96 0.1-2.0 Arginine 4.42 2.0-6.0Tryptophan 0.95 0.1-2.0 Total 41.69 11.3-70 

Example 3

Samples of mixotrophically cultured Chlorella whole cells were analyzedfor carbohydrate content. The results of the sample analysis andextrapolated ranges are shown in Tables 6-7.

TABLE 6 % in Range (% in Analyte Carbohydrates % in Biomass biomass)Polysaccharide 81.61 32.6 20-40 Raffinose 1.47 0.6 0.1-2.0 Cellobiose1.89 0.8 0.1-2.0 Maltose 5.18 2.1 0.1-4.0 Glucose 5 2 0.1-4.0 Xylose 0.70.3 0.1-1.0 Galactose 1.21 0.5 0.1-1.0 Mannose 0.86 0.3 0.1-1.0 Fructose0.41 0.2 0.1-1.0 Glucuronic acid 1.67 0.7 0.1-2.0 Total 100 40.120.9-58.0

TABLE 7 % in Range (% in Analyte Carbohydrates % in Biomass Biomass)Glucose 54.5 21.8 10-30 Xylose 4.5 1.8 0.1-4  Galactose 16.5 6.6 4.0-8.0Arabinose 5.2 2.1 0.1-4.0 Mannose 5.6 2.2 0.1-4.0 Fructose 2.7 1.10.1-2.0 Glucuronic acid 10 4 2.0-6.0 Total 99 39.6 16.4-58.0

Example 4

Samples of a low concentration mixotrophic Chlorella based compositioncomprising 10% by weight mixotrophic Chlorella whole cells, <0.1%phosphoric acid, 0.3% potassium sorbate, and the remaining balance ofwater were analyzed for content. The results of the sample analysis andextrapolated ranges based on standard deviations are shown in Table 8,with NA indicating levels that were too low for detection. The resultsof the protein analysis are presented on a dry weight basis, while theremaining results are presented on a wet basis.

TABLE 8 Sample No. 1 2 3 4 Range % Protein (Leco) 31.1 28.7 23.4 2217-35 % Lipids (AOAC) 10.12 8.82 13.15 12.2  6-16 Nitrogen (ppm) 49764592 3744 3520 3,000-7,000 Phosphorus (ppm) 1600 1300 1500 14001,200-1,700 Potassium (ppm) 979.4 961.8 1385.5 1319.6  700-1700 Boron(ppm) NA NA NA NA Calcium (ppm) 160 100 120 130  65-200 Iron (ppm) 119.9 9.6 9.3  8-12 Magnesium (ppm) 130 94 95 86  70-160 Manganese (ppm)2.5 2.0 2.1 1.8 1.5-3.0 Sulfur (ppm) NA NA NA NA Zinc (ppm) NA NA NA NAArsenic (ppm) NA NA NA NA Cadmium (ppm) NA NA NA NA Cobalt (ppm) 1.2 ×10⁻⁵ 1.1 × 10⁻⁵ 1.1 × 10⁻⁵ 1.2 × 10⁻⁵  0.00001-0.000013 Chromium (ppm)NA NA NA NA Copper (ppm) 5.5 × 10⁻⁴ 2.5 × 10⁻⁴ NA 3.9 × 10⁻⁴0.00002-0.00006 Mercury (ppm) NA NA NA NA 0.1-2.0 Molybedenum NA NA NANA 0.1-2.5 (ppm) Sodium (ppm) 0.047 0.028 0.028 0.022 0.017-0.058 Nickel(ppm) NA NA NA NA Lead (ppm) NA NA NA NA Selenium (ppm) NA NA NA NAAerobic Plate 380,000 130,000 91,000 56,000  80,000-400,000 Count FSNS #1.1 (FDA-BAM) (Est CFU/mL) Salmonella FSNS # (—) 25 (—) 25 (—) 25 (—) 250 32.2 (ELFA- gram gram gram gram AOAC) Staphylococcus <10 <10 <10 <100.1-10  aureus FSNS # 11.1 (FDA-BAM) (CFU/mL) Coliform Count <3.0 <3.0<3.0 <3.0 0.1-3.0 MPN FSNS # 7.1 (FDA-BAM) (MPN/mL) E. coli MPN FSNS<3.0 <3.0 <3.0 <3.0 0.1-3.0 # 7.1 (FDA-BAM) (MPN/mL) Mold Count FSNS <10<10 <10 <10 0.1-10  # 4.1 (FDA-BAM) (CFU/mL) Yeast Count FSNS <10 <10 10<10 0.1-15  # 4.1 (FDA-BAM) (CFU/mL)

Example 5

Samples of mixotrophic Chlorella whole cells and low concentrationmixotrophic Chlorella based compositions comprising 10% by weightmixotrophic Chlorella whole cells, <0.1% phosphoric acid, 0.3% potassiumsorbate, and the remaining balance of water were analyzed by theNational Research Council Canada (Ottawa, Ontario) for phytohormonecontent. The mixotrophic Chlorella based compositions used in thisExample were not analyzed to quantify bacteria in the compositions,however aerobic plate counts for previous compositions prepared with thesame components in the same manner contained 40,000-400,000 CFU/mL. Allmixotrophic Chlorella whole cell samples had to be dried for analysis,and the results are reported with respect to dry weight (DW). Twosamples of mixotrophic Chlorella whole cells analyzed containedmixotrophic Chlorella which had been dried by a drum drier prior toanalysis, consisting of one sample where the mixotrophic Chlorella wholecells had been previously stored in a freezer (old) and one sample wherethe mixotrophic Chlorella whole cells had not been previously stored(fresh). A sample of mixotrophic Chlorella whole cells which was freezedried before analysis was used as the closest approximation of thecontent of mixotrophic Chlorella cells that have not been subjected to adrying process. Samples of dried phototrophically cultured Chlorellavulgaris was obtained from Hoosier Hill Farm LLC (Angola, Ind.).

The low concentration mixotrophic Chlorella based composition sampleswere analyzed as liquid samples, and the results are reported withrespect to fresh weight (FW). One sample contained mixotrophic Chlorellabased composition that had been previously stored in a freezer (old) andone sample contained mixotrophic Chlorella based composition that hadnot been previously stored (fresh). The results of the sample analysisare shown in Tables 9-12, with n.d. indicated where the metabolite wasnot detected. The reported ng/g is equivalent to parts per billion (ppb)levels.

TABLE 9 ABA and ABA metabolites (ng/g DW) Solid Sample ABA ABAGE PANeo-PA t-ABA Phototrophic Chlorella <4 n.d. <4 n.d. <4 vulgarisMixotrophic Chlorella 8 n.d n.d <3.9 11 sp. - drum dried (stored)Mixotrophic Chlorella <3.9 <3.9   <3.9 n.d. <3.9 sp. - Drum Dried(fresh) Mixotrophic Chlorella 11 <3.9  7 <3.9 15 sp. - Freeze Dried(stored) ABA and ABA metabolites (ng/g FW) Liquid Sample ABA ABAGE PANeo-PA t-ABA Stored Mixotrophic <0.4 n.d. n.d. n.d. n.d. Chlorellacomposition Fresh Mixotrophic n.d. <0.4 n.d. n.d. <0.4 Chlorellacomposition

The phytohormones in Table 9 are abbreviated as follows:ABA=cis-Abscisic acid; ABAGE=Abscisic acid glucose ester; PA=Phaseicacid; Neo-PA=Neo-Phaseic acid; and t-ABA=trans-Abscisic acid. As shownin Table 9, both drum dried samples showed lower levels of ABA and ABAmetabolites than the freeze dried sample. The mixotrophic Chlorellacells showed comparable levels of ABA and ABA metabolites to thephototrophic Chlorella cells samples. Neither of the low concentrationmixotrophic Chlorella based composition samples showed detectable levelsof ABA and ABA metabolites.

TABLE 10 Cytokinins (ng/g DW) Solid Sample t-ZOG t-Z c-Z t-ZR c-ZR dhZRiP iPR Phototrophic n.d. n.d. 2 <1 12 4 3 5 Chlorella vulgarisMixotrophic n.d. <1.3 7 17 238 n.d. 3 13 Chlorella sp. - drum dried(stored) Mixotrophic n.d. n.d. <1.2 6 233 1 <1 4 Chlorella sp. - DrumDried (fresh) Mixotrophic n.d. 3  14 11 42 <1  6 3 Chlorella sp. -Freeze Dried (stored) Cytokinins (ng/g FW) Liquid Sample t-ZOG t-Z c-Zt-ZR c-ZR dhZR iP iPR Stored Mixotrophic n.d. n.d. 0 <0.1 13 n.d. <0.10.4 Chlorella composition Fresh Mixotrophic 2 n.d. 14 n.d. 6 n.d. 4 1Chlorella composition

The phytohormones in Table 10 are abbreviated as follows: t-ZOG=(trans)Zeatin-O-glucoside; t-Z=(trans) Zeatin; c-Z=(cis) Zeatin; t-ZR=(trans)Zeatin riboside; c-ZR=(cis) Zeatin riboside; dhZR=Dihydrozeatinriboside; iP=Isopentenyladenine; and iPR=Isopentenyladenosine. As shownin Table 10, both drum dried samples showed lower levels of t-Z, c-Z,and iP than the freeze dried sample. The composition samples showeddetectable levels of t-ZOG, c-Z, c-ZR, iP, and iPR, indicating thatsubjecting the mixotrophic Chlorella based composition to a drum dryingprocess can reduce the c-Z and iP content of the composition. Themixotrophic Chlorella cells samples showed higher content of t-ZR thanthe phototrophic Chlorella cells sample. The low concentrationmixotrophic Chlorella based composition samples showed detectable levelsof t-ZOG, c-Z, c-ZR, iP, and iPR.

TABLE 11 Auxins (ng/g DW) IAA- IAA- IAA- IAA- Solid Sample IAA Ala AspGlu Leu Phototrophic Chlorella 70 n.d. <4 n.d. n.d. vulgaris MixotrophicChlorella 412 n.d. n.d. n.d. n.d. sp. - drum dried (stored) MixotrophicChlorella 414 <3.9 n.d. n.d. n.d. sp. - Drum Dried (fresh) MixotrophicChlorella 794 n.d. n.d. n.d. n.d. sp. - Freeze Dried (stored) Auxins(ng/g FW) IAA- IAA- IAA- IAA- Liquid Sample IAA Ala Asp Glu Leu StoredMixotrophic 2 n.d. n.d. n.d. n.d. Chlorella composition FreshMixotrophic 27 n.d. n.d. n.d. n.d. Chlorella composition

The phytohormones in Table 11 are abbreviated as followsIAA=Indole-3-acetic acid; IAA-Ala=N-(Indole-3-yl-acetyl)-alanine;IAA-Asp=N-(Indole-3-yl-acetyl)-aspartic acid;IAA-Glu=N-(Indole-3-yl-acetyl)-glutamic acid; andIAA-Leu=N-(Indole-3-yl-acetyl)-leucine. As shown in Table 11, both drumdried samples showed lower levels of IAA than the freeze dried sample,and the mixotrophic Chlorella cells samples showed IAA levels higherthan the phototrophic Chlorella cells samples. The composition samplesshowed detectable levels of IAA, indicating that subjecting themixotrophic Chlorella based composition to a drum drying process canreduce the IAA content of the composition.

TABLE 12 Gibberellins (ng/g DW) Solid Sample GA3 GA4 GA7 GA8 GA34 GA44GA51 GA53 Phototrophic <4 <4   n.d. n.d. n.d. <4   n.d. n.d. Chlorellavulgaris Mixotrophic <3.9 n.d. n.d. n.d. n.d. n.d. n.d. n.d. Chlorellasp. - drum dried (stored) Mixotrophic <3.9 n.d. n.d. n.d. n.d. <3.9 n.d.n.d. Chlorella sp. - Drum Dried (fresh) Mixotrophic 7 n.d. n.d. n.d.<3.9 n.d. n.d. n.d. Chlorella sp. - Freeze Dried (stored) Gibberellins(ng/g FW) Liquid Sample GA3 GA4 GA7 GA8 GA34 GA44 GA51 GA53 StoredMixotrophic n.d. <0.4 n.d. n.d. <0.4 n.d. n.d. n.d. Chlorellacomposition Fresh Mixotrophic n.d. <0.4 n.d. n.d. n.d. n.d. n.d. n.d.Chlorella composition

The phytohormones in Table 12 are abbreviated as follows:GA=Gibberellins. As shown in Table 12, both drum dried samples showedlower levels of GA3 than the freeze dried sample. Neither of thecomposition samples showed detectable levels of Gibberellins.

Example 6

An experiment was conducted to determine if application of a lowconcentration of a mixotrophic Chlorella based composition to tomatoseeds planted in soil affected the rate at which the seedlings emergefrom the soil. Tomatoes are part of the Solanaceae family. Tomato seeds(Solanum lycopersicum) were planted in trays with standard soillessplant potting soil mix. Ten treatments were compared to an untreatedcontrol (UTC) and are listed in Table 13, with treatments 3 and 9 beingduplicates. The treatments consisted of one treatment where themixotrophic Chlorella based composition had been dried by a drum drier(DD) before formulation for treatment, and two treatments where themixotrophic Chlorella based composition had not been dried (wet). Themixotrophic Chlorella based composition in treatments 3 and 9 was notsubjected to a drying or lysing process. The Haematococcus pluvialisextracted biomass was mechanically lysed before being subjected to asupercritical carbon dioxide extraction process. The mixotrophicallycultured Galidieria sp. lysed cells were mechanically lysed. The BG-11culture media treatment consisted of the same culture media used in themixotrophic Chlorella culturing process. The centrifuged media treatmentconsisted of the cultured media separated from a mixotrophic Chlorellaculture by centrifuge at the end of the culturing process (i.e., oncethe mixotrophic Chlorella was harvested). A commercially availablemacroalgae extract based product was obtained from Acadian SeaplantsLimited (30 Brown Avenue, Dartmouth, Nova Scotia, Canada, B3B 1X8) forcomparison. The commercially available product Transit Soil fromFBSciences, Inc. (153 N Main Street, Ste 100, Collierville, Tenn. 38017)was also tested.

TABLE 13 Treatment No. Treatment Description 1 UTC - untreated watercheck 2 Mixotrophic Chlorella sp. - Drum Dried Whole Cells (DD) 3Mixotrophic Chlorella sp. - Whole Cells (Wet Plot 1) 4 PhototrophicHaematococcus pluvialis - Extracted Biomass 5 Mixotrophic Galdieriasp. - Whole Cells 6 Mixotrophic Galdieria sp. - Lysed Cells 7Centrifuged Media from Mixotrophic Chlorella sp. culture 8 BG-11 CultureMedia 9 Mixotrophic Chlorella sp. - Whole Cells (Wet Plot 2) 10 GrowerStandard Product - Acadian Liquid Seaweed Concentrate 11 Grower StandardProduct - Transit Soil

The treatments were pasteurized, normalized to 10% solids (fortreatments with microalgal solids), and stabilized with phosphoric acid(H₃PO₄) and potassium sorbate (C₆H₇KO₂), with the remaining balanceconsisting of water. The mixotrophic Chlorella based compositions werepreviously frozen and thawed, and were incorporated into the formulatedtreatments used in this experiment after cold storage following beingharvested from the microalgae culturing system. The mixotrophicChlorella based compositions used in the treatments of this experimentwere not analyzed to quantify bacteria in the compositions, howeveraerobic plate counts for previous compositions prepared with the samecomponents in the same manner contained 40,000-400,000 CFU/mL.

All treatments were applied to the seeds at the low concentration of4.73 mL/gallon. The treatment method consisted of drenching the soil ata rate of 100 gallons/acre using a watering can. The treatments wereapplied immediately after planting the seeds. The tested concentrationof 4.73 mL/gallon diluted the composition which originally contained 10%solids by weight of mixotrophic Chlorella whole cells to the low percentsolids content of only 0.012495%.

Each treatment was applied to 100 seeds planted in a 10 by 10 pattern inplanting trays, with each row of ten counting as a replicate (10 totalreplicates). Visual observations were made daily to record thepercentage of plants that have emerged from the soil. The standard usedfor assessing emergence was the hypocotyl stage where a stem was visibleto be protruding from the potting soil mix. The experiment was conductedinside a greenhouse with all seeds and treatments subjected to the samecontrolled conditions including temperature and light. All trays weretreated with the same amount of water throughout the experiment. Noadditional nutrients were provided to the plants during the experiment.All data rated as significant was done so utilizing the New Duncan'sMultiple Test Range at a 90% confidence level, such that values with astatistical significant identifier of the same letter are notsignificantly different.

Results are shown in Table 14-18 with accompanying statisticalsignificance grouping identifiers.

TABLE 14 Plant Emergence (Ave. % of plants emerged on date) Day 1 Day 2Day 3 AM PM AM PM AM PM 1 0 a 0 c 0 d 2 f 3 d 16 d 2 0 a 0 c 0 d 1 f 2 d21 d 3 0 a 3 c 6 c 24 d 23 bcd 60 b 4 0 a 3 c 4 c 24 d 26 bcd 60 b 5 0 a0 c 0 d 5 f 6 d 45 c 6 0 a 0 c 0 d 5 f 5 d 44 c 7 0 a 0 c 0 d 7 f 10 d43 c 8 0 a 0 c 0 d 10 ef 10 d 49 bc 9 0 a 8 ab 10 ab 42 b 45 ab 72 a 100 a 0 c 0 d 18 de 19 cd 6 b 11 0 a 0 c 0 d 16 de 44 ab 44 c

TABLE 15 Plant Emergence (Ave. % of plants emerged on date) Day 4 Day 5Day 6 Day 7 AM PM AM PM PM PM 1 17 g 47 g 55 e 76 a 83 a 84 A 2 24 g 55fg 56 e 77 a 84 a 87 A 3 65 abc 70 b-e 79 abc 83 a 82 a 78 A 4 61 bcd 73a-e 79 abc 84 a 84 a 85 A 5 44 ef 64 def 64 de 82 a 83 a 88 A 6 43 f 61ef 66 cde 77 a 80 a 80 A 7 44 ef 64 def 73 a-d 82 a 81 a 83 A 8 56 cde64 def 66 cde 77 a 74 a 77 a 9 73 ab 80 ab 83 ab 85 a 88 a 87 a 10 62bcd 79 abc 79 abc 85 a 89 a 88 a 11 47 ef 68 cde 72 a-d 79 a 83 a 84 a

As shown in Tables 14-15, treatments 3 and 9 comprising the mixotrophicChlorella based composition emerged out of the soil sooner than the UTC,the grower standard commercial products in treatments 10 and 11, andtreatments 5-8, showing a statistically significant difference on Day 2AM. The percentage of plants emerged for all treatments converged at theend of the experiment.

TABLE 16 Plant Emergence (Ave. % of plants emerged at observation time)Un- treated Mixotrophic Mixotrophic Water Chlorella sp. % In- Chlorellasp. % In- Check Whole Cells crease Whole Cells crease (UTC) (Treatment3) over UTC (Treatment 9) over UTC Day  0 a  0 a  0 a 1 AM Day  0 c  3 c 8 ab 1 PM Day  0 d  6 d  10 ab 2 AM Day  2 f 24 d 1100%  42 b 2000%  2PM Day  3 d  23 bcd 667%  45 ab 1400%  3 AM Day 16 d 60 b 275% 72 a350%  3 PM Day 17 g  65 abc 282%  73 ab 329%  4 AM Day 47 g   70 b-e 49%  80 ab 70% 4 PM Day 55 e  79 abc  44%  83 ab 51% 5 AM Day 76 a 83 a 9% 85 a 12% 5 PM Day 83 a 82 a  −1% 88 a  6% 6 PM Day 84 a 78 a  −7% 87a  4% 7 PM

Table 16 shows treatments 3 and 9 comprising the mixotrophic Chlorellabased composition with respect to the UTC. As shown in Table 16,treatments 3 and 9 began emerging from the soil on Day 1 PM, while theUTC treatment did not begin emergence until Day 2 PM and lagged behindtreatments 3 and 9 by a statistically significant margin on most daysuntil Day 5 PM. Of the plots receiving treatments comprising themixotrophic Chlorella based composition, treatment 3 demonstrated astatistically significant difference from the UTC at Day 2 PM, Day 3 PM,Day 4 AM, Day 4 PM and Day 5 AM, and treatment 9 demonstrated astatistically significant difference from the UTC from Day 1 PM throughDay 5 AM. Treatments 3 and 9 also reached at least 70% emergence a daybefore the UTC, and maintained a numerical increase of at least 27% overthe UTC through Day 5 AM.

TABLE 17 Plant Emergence (Ave. % of plants emerged at observation time)Un- Mixotrophic Mixotrophic treated Mixotrophic Chlorella Wet ChlorellaWet Control Chlorella DD Plot 1 Plot 2 (UTC) (Treatment 2) (Treatment 3)(Treatment 9) Day 1 AM  0 a  0 a  0 a  0 a Day 1 PM  0 c  0 c  3 c  8 abDay 2 AM  0 d  0 d  6 d  10 ab Day 2 PM  2 f  1 f 24 d 42 b % over UTC−50%  1100%  2000%  % over DD 2300%  4100%  Day 3 AM  3 d  2 d  23 bcd 45 ab % over UTC −33%  667%  1400%  % over DD 1050%  2150%  Day 3 PM 16d 21 d 60 b 72 a % over UTC 31%  275%  350%  % over DD 186%  242%  Day 4AM 17 g 24 g  65 abc  73 ab % over UTC 41%  282%  329%  % over DD 171% 204%  Day 4 PM 47 g  55 fg   70 b-e  80 ab % over UTC 17%  49% 70% %over DD 27% 45% Day 5 AM 55 e 56 e  79 abc  83 ab % over UTC 2% 44% 51%% over DD 41% 48% Day 5 PM 76 a 77 a 83 a 85 a % over UTC 1%  9% 12% %over DD  8% 10% Day 6 PM 83 a 84 a 82 a 88 a % over UTC 1% −1%  6% %over DD −2%  5% Day 7 PM 84 a 87 a 78 a 87 a % over UTC 4% −7%  4% %over DD −10%   0%

As shown in Table 17, the two treatments comprising wet mixotrophicChlorella based composition emerged from the soil faster than the UTCand treatment comprising DD mixotrophic Chlorella based composition. Ofthe plots receiving treatments comprising wet mixotrophic Chlorellabased composition, the first plot demonstrated a statisticallysignificant difference from the UTC and treatment comprising DDmixotrophic Chlorella based composition at Day 2 PM, Day 3 PM, Day 4 AM,Day 4 PM and Day 5 AM, and the second plot demonstrated a statisticallysignificant difference from the UTC and treatment comprising DDmixotrophic Chlorella based composition from Day 1 PM through Day 5 AM.The treatments comprising wet mixotrophic Chlorella based compositionalso reached at least 70% emergence a day before the UTC and treatmentcomprising DD mixotrophic Chlorella based composition, and maintained anumerical increase of at least 27% over the UTC and treatment comprisingDD Chlorella based composition through Day 5 AM. The performance of thetreatment comprising DD mixotrophic Chlorella based composition largelymirrored the performance of the UTC, with no statistically significantdifference over the course of the experiment and numerical increasesabove 10% on only Day 3 PM through Day 4 PM. Thus the results indicatethat drying the mixotrophic Chlorella based composition with a drumdrier in the preparation process reduced the effectiveness of thecompositions to accelerate emergence of the tomato plants when appliedas soil drench.

TABLE 18 Plant Emergence (Ave. % of plants emerged on Day 4 AM) % 22-Mayincrease AM over UTC UTC - untreated water check 17 f MixotrophicChlorella sp. - Drum Dried 24 f  41% Whole Cells (DD) MixotrophicChlorella sp. - Whole Cells 69 ab 306% (Average of Wet Plots 1 and 2)Phototrophic Haematococcus pluvialis - 61 bcd 259% Extracted BiomassMixotrophic Galdieria sp. - Whole Cells 44 e 159% Mixotrophic Galdieriasp. - Lysed Cells 43 e 153% Centrifuged Media from Mixotrophic 44 e 159%Chlorella sp. culture BG-11 Culture Media 56 cde 229% Grower StandardProduct - Acadian 62 abc 265% Liquid Seaweed Concentrate Grower StandardProduct - Transit Soil 47 de 176%

Table 18 displays the data from the Day 4 AM with the duplicatemixotrophic Chlorella based composition treatments averaged forcomparison to the other treatments, and shows a statisticallysignificant difference for the mixotrophic Chlorella based compositionwhich had not been dried (i.e., wet) as compared to the UTC, whichamounts to a numerical increase of 306%. Table 18 also shows that themixotrophic Chlorella based composition treatment that had not beendried outperformed the commercially available products, and wassignificantly different from the mixotrophic Galdieria and drum driedmixotrophic Chlorella based composition treatments.

Example 7

An experiment was conducted to determine if the method of application ofa low concentration of a mixotrophic Chlorella based composition totomato seeds planted in soil affected the rate at which the seedlingsemerge from the soil and mature. Tomato seeds (Solanum lycopersicum)were planted in trays with a potting soil mix of sphagnum moss, perlite,and vermiculite (2:1:1). Three treatments comprising a mixotrophicChlorella based composition were compared to an untreated control (UTC).The treatments were pasteurized, normalized to 10% solids, andstabilized with phosphoric acid (H₃PO₄) and potassium sorbate (C₆H₇KO₂),with the remaining balance consisting of water. The stored mixotrophicChlorella based composition was frozen after being harvested from themicroalgae culturing system and thawed before formulation in the liquidcomposition for treatments used in the experiment. The fresh mixotrophicChlorella based composition was not previously frozen, and wasincorporated into the liquid composition for treatments used in thisexperiment directly after being harvested from the microalgae culturingsystem. The composition used in the treatments of this experiment werenot analyzed to quantify bacteria in the compositions, however aerobicplate counts for previous compositions prepared with the same componentsin the same manner contained 40,000-400,000 CFU/mL.

The mixotrophic Chlorella based liquid composition treatments wereapplied to the seeds through two different treatment methods. The firsttreatment method comprised soaking the seeds in the low concentration of8 mL/gallon of the mixotrophic Chlorella based liquid composition fortwo hours with constant sparging of air to avoid oxygen deprivation,removing the seeds from the composition, drying the seeds overnight, andthen planting the seeds in the potting soil mix. The second treatmentmethod comprised soaking the seeds in water for two hours with constantsparing of air to avoid oxygen deprivation, removing the seeds fromwater, drying the seeds overnight, planting the seeds in the pottingsoil mix with the low concentration of 8 mL/gallon of the mixotrophicChlorella based liquid composition in the base of the planting tray toallow the seeds to be treated with the liquid composition throughcapillary action. The tested concentration of 8 mL/gallon diluted thecomposition which originally contained 10% solids by weight ofmixotrophic Chlorella whole cells to the low percent solids content ofonly 0.021134%.

Each of the three treatments were applied to 72 seeds. Visualobservations of the soil and plants were made daily on days 6 and 7 torecord how many seeds had achieved emergence and maturation, asexplained below. The standard used for assessing emergence was theachievement of the hypocotyl stage, where a stem was visibly protrudingfrom the potting soil mix. The standard used for assessing maturationwas the achievement of the cotyledon stage, where two leaves had visiblyformed on the emerged stem. The experiment was conducted indoors withall seeds and treatments subjected to the same controlled conditionsincluding temperature, light, and supply of water. No other nutrientswere supplied during the experiment. Light supplied was artificial andprovided by fluorescent bulbs 24 hours a day. Results of the experimentare presented in Tables 19-24.

TABLE 19 Number of plants emerged by day Day 6 Day 7 Untreated Control(UTC) 28 42 10% Mixotrophic 22 46 Chlorella Fresh Soak 10% Mixotrophic26 47 Chlorella Stored Soak 10% Mixotrophic 43 58 Chlorella FreshCapillary

TABLE 20 % of total plants emerged by day Day 6 Day 7 Untreated Control(UTC) 39 58 10% Mixotrophic 31 64 Chlorella Fresh Soak 10% Mixotrophic36 65 Chlorella Stored Soak 10% Mixotrophic 60 81 Chlorella FreshCapillary

TABLE 21 % increase of plants emerged by day over UTC Day 6 Day 7 10%Mixotrophic −21%  10% Chlorella Fresh Soak 10% Mixotrophic −7% 12%Chlorella Stored Soak 10% Mixotrophic 54% 38% Chlorella Fresh Capillary

As shown in the Tables 19-21, the capillary action treatment and seedsoak treatments showed higher performance by day seven than the UTCregarding emergence of the plants. On day seven the capillary actiontreatment showed an increase of 38%, the seed soak treatment with storedmixotrophic Chlorella based composition showed an increase of 12%, andthe seed soak treatment with fresh mixotrophic Chlorella basedcomposition showed an increase of 10% over the UTC. These results showthat a low concentration of a mixotrophic Chlorella based composition iseffective in increasing the emergence of a seedling as compared to anuntreated seed when applied in a capillary action application.

TABLE 22 Number of plants matured by day Day 6 Day 7 Untreated Control(UTC) 11 37 10% Mixotrophic 18 41 Chlorella Fresh Soak 10% Mixotrophic18 39 Chlorella Stored Soak 10% Mixotrophic 23 47 Chlorella FreshCapillary

TABLE 23 % of total plants matured by day Day 6 Day 7 Untreated Control(UTC) 15 51 10% Mixotrophic 25 57 Chlorella Fresh Soak 10% Mixotrophic25 54 Chlorella Stored Soak 10% Mixotrophic 32 65 Chlorella FreshCapillary

TABLE 24 % increase of plants matured by day over the UTC Day 6 Day 710% Mixotrophic 64% 11% Chlorella Fresh Soak 10% Mixotrophic 64%  5%Chlorella Stored Soak 10% Mixotrophic 109%  27% Chlorella FreshCapillary

As shown in the Tables 22-24, the capillary action treatment and seedsoak treatments showed higher performance on days 6 and 7 than the UTCregarding maturation of the plants. The capillary action treatmentshowed an increase of at least 27%, the seed soak treatment with storedmixotrophic Chlorella based composition showed an increase of at least5%, and the seed soak treatment with fresh mixotrophic Chlorella basedcomposition showed an increase of at least 11% over the UTC. Theseresults show that a low concentration of a mixotrophic Chlorella basedcomposition is effective in increasing the maturation of a seedling ascompared to an untreated seed when applied in a capillary actionapplication.

Example 8

An experiment was conducted to determine if the method of application ofa low concentration of a mixotrophic Chlorella based composition totomato seeds planted in soil affected the rate at which the seedlingsemerge from the soil and mature. Tomato seeds (Solanum lycopersicum)were planted in trays with a potting soil mix of sphagnum moss, perlite,and vermiculite (2:1:1). Two treatments comprising mixotrophicallycultured a mixotrophic Chlorella based composition were compared to anuntreated control (UTC). The treatments were pasteurized, normalized to10% solids, and stabilized with phosphoric acid (H₃PO₄) and potassiumsorbate (C₆H₇KO₂), with the remaining balance consisting of water. Themixotrophic Chlorella based composition was not previously frozen, andwas incorporated into the liquid composition for treatments used in thisexperiment directly after being harvested from the microalgae culturingsystem. The composition used in the treatments of this experiment wasnot analyzed to quantify bacteria in the composition, however aerobicplate counts for previous compositions prepared with the same componentsin the same manner contained 40,000-400,000 CFU/mL.

The mixotrophic Chlorella based liquid composition was applied to theseeds at two different concentrations, 4.7 mL/gallon or 8 mL/gallon,using the same treatment method. The tested concentration of 4.7mL/gallon diluted the composition which originally contained 10% solidsby weight of mixotrophic Chlorella whole cells to the low percent solidscontent of only 0.012416%. The tested concentration of 8 mL/gallondiluted the composition which originally contained 10% solids by weightof mixotrophic Chlorella whole cells to the low percent solids contentof only 0.021134%. The treatment method consisted of drenching the soilfrom the top with 0.75 gallon of the liquid composition (equivalent toan application rate of 100 gallons/acre) at the identifiedconcentrations after planting the seeds.

Each of the two treatments were applied to two trays of 72 seeds. Visualobservations of the soil and plants were made daily to record how manyseeds had achieved emergence and maturation, as explained below. Thestandard used for assessing emergence was the achievement of thehypocotyl stage where a stem was visibly protruding from the pottingsoil mix. The standard used for assessing maturation was the achievementof the cotyledon stage where two leaves had visibly formed on theemerged stem. The experiment was conducted indoors with all seeds andtreatments subjected to the same controlled conditions includingtemperature, light, and supply of water. No other nutrients weresupplied during the experiment. Light supplied was artificial andprovided by fluorescent bulbs 24 hours a day. Results of the experimentare presented in Tables 25-30.

TABLE 25 Number of plants emerged by day 1 2 3 4 5 6 7 8 9 UntreatedControl — — 0 0 0 10 40 43 69 (UTC) 10% Mixotrophic — — 0 0 0 20 57 6287 Chlorella 4.7 mL 10% Mixotrophic — — 0 0 0 39 59 65 88 Chlorella 8 mL

TABLE 26 % of total plants emerged by day 1 2 3 4 5 6 7 8 9 UntreatedControl 0 0 0 0 0 7 28 30 48 (UTC) 10% Mixotrophic 0 0 0 0 0 14 40 43 60Chlorella 4.7 mL 10% Mixotrophic 0 0 0 0 0 27 41 45 61 Chlorella 8 mL

TABLE 27 % increase of plants emerged by day over the UTC 1 2 3 4 5 6 78 9 10% Mixotrophic — — — — — 100% 43% 44% 26% Chlorella 4.7 mL 10%Mixotrophic — — — — — 290% 48% 51% 28% Chlorella 8 mL

As shown in the Tables 25-27, the 8 and 4.7 mL/gallon applicationsshowed consistently higher performance than the UTC regarding emergenceof the plants, with the 8 mL/gallon application consistently performingbetter than the 4.7 mL/gallon. The 4.7 mL/gallon application showed atleast a 26% and as much as a 100% increase over the UTC on comparativedays, and the 8 mL/gallon application demonstrated at least a 28% and asmuch as a 290% increase over the UTC. The largest difference between the4.7 and 8 mL/gallon applications occurred on day 6. These results showthat a low concentration of a mixotrophic Chlorella based composition iseffective in increasing the emergence of a seedling as compared to anuntreated seed when applied in a soil drench application.

TABLE 28 Number of plants matured by day 1 2 3 4 5 6 7 8 9 UntreatedControl — — 0 0 0 2 22 45 65 (UTC) 10% Mixotrophic — — 0 0 0 9 42 69 83Chlorella 4.7 mL 10% Mixotrophic — — 0 0 0 8 46 68 79 Chlorella 8 mL

TABLE 29 % of total plants matured by day 1 2 3 4 5 6 7 8 9 UntreatedControl 0 0 0 0 0 1 15 31 45 (UTC) 10% Mixotrophic 0 0 0 0 0 6 29 48 58Chlorella 4.7 mL 10% Mixotrophic 0 0 0 0 0 6 32 47 55 Chlorella 8 mL

TABLE 30 % increase of plants matured by day over the UTC 1 2 3 4 5 6 78 9 10% Mixotrophic — — — — — 350%  91% 53% 28% Chlorella 4.7 mL 10%Mixotrophic — — — — — 300% 109% 51% 22% Chlorella 8 mL

As shown in the Tables 28-30, the 8 and 4.7 mL/gallon applicationsshowed consistently higher performance than the UTC regarding maturationof the plants, with the 4.7 mL/gallon application performing better thanthe 8 mL/gallon on days 6, 8, and 9. The 4.7 mL/gallon applicationshowed at least a 28% and as much as a 350% increase over the UTC oncomparative days and the 8 mL/gallon application demonstrated at least a22% and as much as a 300% increase over the UTC. These results show thata low concentration of a mixotrophic Chlorella based composition iseffective in increasing the maturation of a seedling as compared to anuntreated seed when applied in a soil drench application.

Example 9

An experiment was conducted to determine if a low concentration and lowfrequency application of mixotrophic Chlorella based composition toheirloom tomato (cv German striped) plants by foliar applicationaffected the initial growth and sizing of the plants. Tomato seeds(Solanum lycopersicum) were planted in trays with standard soillessplant potting soil mix and grown in a nursery greenhouse. Treatments ofa mixotrophic Chlorella based composition and a commercially availablereference product were compared to an untreated control (UTC) and arelisted in Table 31, with duplicate treatments of the mixotrophicChlorella based composition being tested. A commercially availablemacroalgae extract based product was obtained from Acadian SeaplantsLimited (30 Brown Avenue, Dartmouth, Nova Scotia, Canada, B3B 1X8) forcomparison.

TABLE 31 Treatment No. Treatment Description 1 UTC - untreated watercheck 2 Mixotrophic Chlorella sp. - Drum Dried Whole Cells (DD) 3Mixotrophic Chlorella sp. - Whole Cells (Wet Plot 1) 4 MixotrophicChlorella sp. - Whole Cells (Wet Plot 2) 5 Grower Standard Product -Acadian Liquid Seaweed Concentrate

The mixotrophic Chlorella based composition was pasteurized, normalizedto 10% solids, and stabilized with phosphoric acid (H₃PO₄) and potassiumsorbate (C₆H₇KO₂), with remaining balance consisting of water. Themixotrophic Chlorella whole cells were not previously subjected to apurification process to isolate the cells from the microalgae culturingmedium, nor were the cells previously subjected to a drying, extraction,or other process that can lyse or disrupt the cell walls, except asindicated for the drum dried treatment. The composition comprisingmixotrophic Chlorella used in the treatments of this experiment were notanalyzed to quantify bacteria in the compositions, however aerobic platecounts for previous compositions prepared with the same components inthe same manner contained 40,000-400,000 CFU/mL. The mixotrophicChlorella composition was previously frozen and thawed, and wasincorporated into the liquid composition for treatments used in thisexperiment after cold storage following being harvested from themicroalgae culturing system.

The mixotrophic Chlorella based composition treatments were applied tothe plants at a concentration of 4 mL/gallon. The tested concentrationof 4 mL/gallon diluted the composition which originally contained 10%solids by weight of mixotrophic Chlorella whole cells to the low percentsolids content of only 0.010567%. The Acadian treatment was applied toplants at a concentration of 9.46 mL/gallon. The low concentration andlow frequency treatment method consisted of directly spraying thefoliage of the plants a rate of 25 gallons/acre using a spray bottle. Atotal of three applications were applied with the first applicationoccurring three weeks after planting (7-10 days after emergence). Thesecond application was applied five days after the first, and the thirdapplication was applied six days after the second.

Each treatment was applied to a 14 inch by 14 inch planting flatscontaining plants resulting from 25-30 seeds. There were eightreplicates of each treatment. All seeds were planted in a standardsoilless potting plant mix. Each plant analyzed was counted as areplicate with eight replicates considered for each treatmentevaluation. Analysis occurred after the second treatment and after thethird treatment. The chlorophyll content was estimated by SPAD(Soil-Plant Analysis Development) value, a numeric value provided by aMinolta SPAD meter which analyzes the amount of light in a specificlight spectrum passing through a leaf and converts that reading to anumerical value as an indicator of chlorophyll density in the leaf. Theexperiment was conducted inside a greenhouse with all seeds andtreatments subjected to the same controlled conditions includingtemperature and light. All trays were treated with the same amount ofwater throughout the experiment. No additional nutrients were providedto the plants during the experiment. All data rated as significant wasdone so utilizing the New Duncan's Multiple Test Range at a 90%confidence level, such that values with a statistical significantidentifier of the same letter are not significantly different. Resultsare shown in Tables 32-37 designated with an F for foliar application,with accompanying statistical significance grouping identifiers.

Example 10

An experiment was conducted to determine if a low concentration and lowfrequency application of mixotrophic Chlorella based composition toheirloom tomato (cv German striped) plants (Solanum lycopersicum) bysoil application affected the initial growth and sizing of the plants.The soil application trial occurred in the same location, with the sametreatments, and with the same design as the experiment in Example 9.

The mixotrophic Chlorella based composition treatments were applied tothe plants at a low concentration of 4.73 mL/gallon. The testedconcentration of 4.73 mL/gallon diluted the composition which originallycontained 10% solids by weight of mixotrophic Chlorella whole cells tothe low percent solids content of only 0.012495%. The Acadian treatmentwas applied to plants at a concentration of 9.46 mL/gallon. The lowconcentration and low frequency treatment method consisted of drenchingthe soil at a rate of 100 gallons/acre. A total of three treatments wereapplied with the first application occurring two weeks after planting(7-10 days after emergence). The second treatment was applied nine daysafter the first, and the third treatment was applied five days after thesecond. All data rated as significant was done so utilizing the NewDuncan's Multiple Test Range at a 90% confidence level, such that valueswith a statistical significant identifier of the same letter are notsignificantly different. Results are shown in Tables 32-37 designatedwith an S for soil application, with accompanying statisticalsignificance grouping identifiers.

TABLE 32 Nursery Tomato Plant Sizing - Plant Height (inches) IncreaseIncrease over over Avg. UTC DD 1 UTC - untreated water check F 6.00 cdeUTC - untreated water check S 5.85 ab 2 Mixotrophic Chlorella sp. - 6.48ab  8% Whole Cells DD F Mixotrophic Chlorella sp. - 5.53 bcd −5% WholeCells DD S 3 Mixotrophic Chlorella sp. - 5.27 fg −12%  −18%  Whole CellsWet Plot 1 F Mixotrophic Chlorella sp. - 5.20 def −11%  −6% Whole CellsWet Plot 1 S 4 Mixotrophic Chlorella sp. - 6.13 abcd  2% −5% Whole CellsWet Plot 2 F Mixotrophic Chlorella sp. - 5.52 bcd −6%  0% Whole CellsWet Plot 2 S 5 Grower Standard Product - 5.94 de −1% Acadian LiquidSeaweed Concentrate F Grower Standard Product - 5.67 abc −3% AcadianLiquid Seaweed Concentrate S

As shown in Table 32, the treatments comprising wet mixotrophicChlorella based composition did not show a statistically significant ornumerical increase over the UTC regarding plant height. Additionally thewet mixotrophic Chlorella based composition did not show a statisticallysignificant or numerical increase over the DD mixotrophic Chlorellabased composition treatment.

TABLE 33 Nursery Tomato Plant Sizing - Leaf Number Increase Increaseover over Avg. UTC DD 1 UTC - untreated water check F 5.1 a UTC -untreated water check S 4.5 a 2 Mixotrophic Chlorella sp. - 5.2 a 2%Whole Cells DD F Mixotrophic Chlorella sp. - 4.6 a 3% Whole Cells DD S 3Mixotrophic Chlorella sp. - 4.9 a −2%  −6% Whole Cells Wet Plot 1 FMixotrophic Chlorella sp. - 4.5 a 0% −2% Whole Cells Wet Plot 1 S 4Mixotrophic Chlorella sp. - 5.3 a 4%  2% Whole Cells Wet Plot 2 FMixotrophic Chlorella sp. - 4.4 a −3%  −4% Whole Cells Wet Plot 2 S 5Grower Standard Product - 4.9 a −2%  Acadian Liquid Seaweed ConcentrateF Grower Standard Product - 4.6 a 1% Acadian Liquid Seaweed ConcentrateS

As shown in Table 33, the treatments comprising the wet mixotrophicChlorella based composition did not show a statistically significantover the UTC or DD mixotrophic Chlorella based composition treatmentregarding leaf number.

TABLE 34 Nursery Tomato Chlorophyll Content (SPAD) Increase Increaseover over Avg. UTC DD 1 UTC - untreated water check F 25.9 f UTC -untreated water check S 30.4 a 2 Mixotrophic Chlorella sp. - 27.8 ef 7%Whole Cells DD F Mixotrophic Chlorella sp. - 29.1 a −4%  Whole Cells DDS 3 Mixotrophic Chlorella sp. - 32.1 bcd 24%  15% Whole Cells Wet Plot 1F Mixotrophic Chlorella sp. - 30.7 a 1%  5% Whole Cells Wet Plot 1 S 4Mixotrophic Chlorella sp. - 34.0 ab 31%  22% Whole Cells Wet Plot 2 FMixotrophic Chlorella sp. - 32.7 a 8% 12% Whole Cells Wet Plot 2 S 5Grower Standard Product - 34.5 ab 33%  Acadian Liquid SeaweedConcentrate F Grower Standard Product - 30.6 a 1% Acadian Liquid SeaweedConcentrate S

As shown in Table 34, the foliar treatments comprising the wetmixotrophic Chlorella based composition did show a statisticallysignificant increase over the UTC and DD mixotrophic Chlorella basedcomposition treatment regarding chlorophyll content. The foliartreatments also showed numerical increases over the UTC of 24% and 31%,as well as numerical increases over the DD mixotrophic Chlorella basedcomposition treatment of 15% and 22%. These results show that smallamounts of the mixotrophic Chlorella based composition at a lowconcentration and low frequency application are effective at improvingchlorophyll content in plants when applied to the foliage. The resultsalso indicate that drying the mixotrophic Chlorella based compositionwith a drum drier in the preparation process reduced the effectivenessof the compositions to enhance the chlorophyll content of the tomatoplants when applied in a foliar application. The soil applicationscomprising the wet mixotrophic Chlorella based composition did not showa statistically significant or numerical increase over the UTC or DDChlorella based composition treatment.

TABLE 35 Nursery Tomato Plant Sizing - Whole Plant Weight (grams)Increase Increase over over Avg. UTC DD 1 UTC - untreated water check F6.8 d UTC - untreated water check S 7.1 a 2 Mixotrophic Chlorella sp. -9.2 ab 36% Whole Cells DD F Mixotrophic Chlorella sp. - 6.3 abc −11% Whole Cells DD S 3 Mixotrophic Chlorella sp. - 6.2 d −8% −33% WholeCells Wet Plot 1 F Mixotrophic Chlorella sp. - 5.3 cdefg −26%  −16%Whole Cells Wet Plot 1 S 4 Mixotrophic Chlorella sp. - 10.6 ab 56%  15%Whole Cells Wet Plot 2 F Mixotrophic Chlorella sp. - 6.5 ab −8%  3%Whole Cells Wet Plot 2 S 5 Grower Standard Product - 8.9 abc 31% AcadianLiquid Seaweed Concentrate F Grower Standard Product - 4.6 efgh −35% Acadian Liquid Seaweed Concentrate S

As shown in Table 35, the foliar treatment comprising wet mixotrophicChlorella based composition in plot 2 did show a statisticallysignificant increase over the UTC and a numerical increase of 56%regarding whole plant weight. The foliar application of the Acadianproduct performed lower, showing only a 31% increase over the UTC. Thefoliar treatment comprising the wet mixotrophic Chlorella basedcomposition in plot 2 did not show a statistically significantdifference over the DD mixotrophic Chlorella based compositiontreatment, but did show a numerical increase of 15%. The foliartreatment in plot 1 and the soil applications comprising wet mixotrophicChlorella based composition did not show a statistically significantincrease over the UTC or DD mixotrophic Chlorella based compositiontreatment. These results show that small amounts of the mixotrophicChlorella based composition at a low concentration and low frequencyapplication are effective at improving whole plant weight when appliedto the foliage.

TABLE 36 Nursery Tomato Plant Sizing - Root Weight (grams) IncreaseIncrease over over Avg. UTC DD 1 UTC - untreated water check F 2.2 bcUTC - untreated water check S 2.8 a 2 Mixotrophic Chlorella sp. - 3.5 a 57% Whole Cells DD F Mixotrophic Chlorella sp. - 2.4 ab −11% WholeCells DD S 3 Mixotrophic Chlorella sp. - 1.9 c −12% −46% Whole Cells WetPlot 1 F Mixotrophic Chlorella sp. - 2.1 bc −24% −13% Whole Cells WetPlot 1 S 4 Mixotrophic Chlorella sp. - 3.3 a  51%  −6% Whole Cells WetPlot 2 F Mixotrophic Chlorella sp. - 1.9 cd −30% −21% Whole Cells WetPlot 2 S 5 Grower Standard Product - 2.8 ab  28% Acadian Liquid SeaweedConcentrate F Grower Standard Product - 1.5 ef −47% Acadian LiquidSeaweed Concentrate S

As shown in Table 36, the foliar application of the mixotrophicChlorella based composition in treatment 4 (wet plot 2) resulted in asignificant difference from the UTC regarding root weight, showing anincrease of 51% over the UTC. The foliar application of the drum driedmixotrophic Chlorella based composition also resulted in a significantdifference from the UTC, with a numerical increase of 57%. The foliarapplication of the Acadian product performed lower, showing only a 28%increase over the UTC. These results show that small amounts of themixotrophic Chlorella based composition at a low concentration and lowfrequency application are effective at improving root weight in plantswhen applied to the foliage.

TABLE 37 Nursery Tomato Plant Sizing - Shoot Weight (grams) IncreaseIncrease over over Avg. UTC DD 1 UTC - untreated water check F 4.6 cdeUTC - untreated water check S 4.3 a 2 Mixotrophic Chlorella sp. - 5.9 bc29% Whole Cells DD F Mixotrophic Chlorella sp. - 3.9 abc −10%  WholeCells DD S 3 Mixotrophic Chlorella sp. - 4.3 e −6% −27% Whole Cells WetPlot 1 F Mixotrophic Chlorella sp. - 3.2 cde −27%  −18% Whole Cells WetPlot 1 S 4 Mixotrophic Chlorella sp. - 7.3 a 60%  24% Whole Cells WetPlot 2 F Mixotrophic Chlorella sp. - 4.6 a  6%  18% Whole Cells Wet Plot2 S 5 Grower Standard Product - 6.1 ab 33% Acadian Liquid SeaweedConcentrate F Grower Standard Product - 3.1 cde −28%  Acadian LiquidSeaweed Concentrate S

As shown in Table 37, the foliar treatment comprising the wetmixotrophic Chlorella based composition in treatment 4 (wet plot 2)showed a statistically significant increase over the UTC and a numericalincrease of 60% regarding shoot weight. The Acadian product performedlower, showing only a 33% increase over the UTC in the foliarapplication, and showing a 28% decrease compared to the UTC in the soilapplication. The foliar treatment comprising the wet mixotrophicChlorella based composition in treatment 4 also showed a statisticallysignificant difference over the DD mixotrophic Chlorella basedcomposition treatment and a numerical increase of 24%. Thus the resultsindicate that drying the mixotrophic Chlorella based composition with adrum drier in the preparation process reduced the effectiveness of thecompositions to enhance the shoot weight of the tomato plants whenapplied in a foliar application. The foliar application in treatment 3(wet plot 1) and the soil applications comprising the wet mixotrophicChlorella based composition did not show a statistically significantincrease over the UTC or DD mixotrophic Chlorella based compositiontreatment, however the soil application in treatment 4 showed an 18%increase over the DD mixotrophic Chlorella based composition treatment.These results show that small amounts of the mixotrophic Chlorella basedcomposition at a low concentration and low frequency application areeffective at improving shoot weight in plants when applied to thefoliage.

Example 11

An experiment was conducted to determine if the method of application ofa low concentration of a mixotrophic Chlorella based composition togreen bean seeds (Phaseolus vulgaris) planted in soil affected the rateat which the seedlings emerge from the soil and mature. Green beans arepart of the Fabaceae family. Green bean seeds were planted in trays witha potting soil mix of sphagnum moss, perlite, and vermiculite (2:1:1).Three treatments comprising a mixotrophic Chlorella based compositionwere compared to an untreated control (UTC). The treatments werepasteurized, normalized to 10% solids, and stabilized with phosphoricacid (H₃PO₄) and potassium sorbate (C₆H₇KO₂), with the remaining balanceconsisting of water. The stored mixotrophic Chlorella based compositionwas frozen after being harvested from the microalgae culturing systemand thawed before formulation in the liquid composition for treatmentsused in the experiment. The fresh mixotrophic Chlorella basedcomposition was not previously frozen, and was incorporated into theliquid composition for treatments used in this experiment directly afterbeing harvested from the microalgae culturing system. The compositionused in the treatments of this experiment were not analyzed to quantifybacteria in the compositions, however aerobic plate counts for previouscompositions prepared with the same components in the same mannercontained 40,000-400,000 CFU/mL.

The mixotrophic Chlorella based liquid composition treatments wereapplied to the seeds through two different treatment methods. The firsttreatment method comprised soaking the seeds in the low concentration of8 mL/gallon of the mixotrophic Chlorella based liquid composition fortwo hours with constant sparging of air to avoid oxygen deprivation,removing the seeds from the composition, drying the seeds overnight, andthen planting the seeds in the potting soil mix. The second treatmentmethod comprised soaking the seeds in water for two hours with constantsparing of air to avoid oxygen deprivation, removing the seeds fromwater, drying the seeds overnight, planting the seeds in the pottingsoil mix with the low concentration of 8 mL/gallon of the mixotrophicChlorella based liquid composition in the base of the planting tray toallow the seeds to be treated with the liquid composition throughcapillary action. The tested concentration of 8 mL/gallon diluted thecomposition which originally contained 10% solids by weight ofmixotrophic Chlorella whole cells to the low percent solids content ofonly 0.021134%.

Each of the three treatments were applied to 72 seeds. Visualobservations of the soil and plants were made daily to record how manyseeds had achieved emergence and maturation, as explained below. Thestandard used for assessing emergence was the achievement of thehypocotyl stage, where a stem was visibly protruding from the pottingsoil mix. The standard used for assessing maturation was the achievementof the cotyledon stage, where two leaves had visibly formed on theemerged stem. The experiment was conducted indoors with all seeds andtreatments subjected to the same controlled conditions includingtemperature, light, and supply of water. No other nutrients weresupplied during the experiment. Light supplied was artificial andprovided by fluorescent bulbs 24 hours a day. Results of the experimentare presented in Tables 38-43.

TABLE 38 Number of plants emerged by day 1 2 3 4 5 6 7 8 UntreatedControl 0 0 0 2 23 30 31 33 (UTC) 10% Mixotrophic 0 0 0 10 36 41 43 45Chlorella Fresh Soak 10% Mixotrophic 0 0 0 3 33 40 42 42 ChlorellaStored Soak 10% Mixotrophic 0 0 0 0 10 15 25 34 Chlorella FreshCapillary

TABLE 39 % of total plants emerged by day 1 2 3 4 5 6 7 8 UntreatedControl 0 0 0 3 32 42 43 46 (UTC) 10% Mixotrophic 0 0 0 14 50 57 60 63Chlorella Fresh Soak 10% Mixotrophic 0 0 0 4 46 56 58 58 ChlorellaStored Soak 10% Mixotrophic 0 0 0 0 14 21 35 47 Chlorella FreshCapillary

TABLE 40 % increase of plants emerged by day over the UTC 1 2 3 4 5 6 78 10% — — —  400% 57% 37% 39% 36% Mixotrophic Chlorella Fresh Soak 10% —— —  50% 43% 33% 35% 27% Mixotrophic Chlorella Stored Soak 10% — — —−100% −57%  −50%  −19%   3% Mixotrophic Chlorella Fresh Capillary

As shown in the Tables 38-40, the seed soak treatment for the fresh andstored mixotrophic Chlorella based compositions showed consistentlyhigher performance than the capillary action treatment and the UTCregarding emergence of the plants. The stored mixotrophic Chlorellabased composition seed soak treatment showed at least a 27% and as muchas a 50% increase over the UTC on comparative days, and the freshmixotrophic Chlorella based composition seed soak treatment demonstratedat least a 36% and as much as a 400% increase over the UTC. Theemergence for the fresh mixotrophic Chlorella based compositionconsistently outperformed the stored mixotrophic Chlorella basedcomposition in the seed soak treatments, with the difference between thetwo treatments being the largest on day 4 and narrowing over theduration of the experiment. These results show that a low concentrationof a mixotrophic Chlorella based composition is effective in increasingthe emergence of a seedling as compared to an untreated seed whenapplied in a seed soak application.

TABLE 41 Number of plants matured by day 1 2 3 4 5 6 7 8 UntreatedControl 0 0 0 0 0 13 21 27 (UTC) 10% Mixotrophic 0 0 0 0 0 25 32 37Chlorella Fresh Soak 10% Mixotrophic 0 0 0 0 0 13 30 35 Chlorella StoredSoak 10% Mixotrophic 0 0 0 0 0 1 6 15 Chlorella Fresh Capillary

TABLE 42 % of total plants matured by day 1 2 3 4 5 6 7 8 UntreatedControl 0 0 0 0 0 18 29 38 (UTC) 10% Mixotrophic 0 0 0 0 0 35 44 51Chlorella Fresh Soak 10% Mixotrophic 0 0 0 0 0 18 42 49 Chlorella StoredSoak 10% Mixotrophic 0 0 0 0 0 1 8 21 Chlorella Fresh Capillary

TABLE 43 % increase of plants matured by day over the UTC 1 2 3 4 5 6 78 10% Mixotrophic — — — — — 92% 52% 37% Chlorella Fresh Soak 10%Mixotrophic — — — — —  0% 43% 30% Chlorella Stored Soak 10% Mixotrophic— — — — — −92%  −71%  −44%  Chlorella Fresh Capillary

As shown in the Tables 41-43, the seed soak treatment for the fresh andstored mixotrophic Chlorella based compositions showed consistentlyhigher performance than the capillary action treatment and the UTCregarding maturation of the plants. The stored mixotrophic Chlorellabased composition seed soak treatment showed at least a 30% and as muchas a 43% increase over the untreated control on comparative days, andthe fresh mixotrophic Chlorella based composition seed soak treatmentdemonstrated at least a 37% and as much as a 92% increase over the UTC.The maturation for the fresh mixotrophic Chlorella compositionconsistently outperformed the stored mixotrophic Chlorella basedcomposition in the seed soak treatments, with the difference between thetwo treatments being the largest on day 6 and narrowing over theduration of the experiment. The capillary action treatment wasconsistently outperformed by the UTC regarding maturation of the plants.These results show that a low concentration of a mixotrophic Chlorellabased composition is effective in increasing the maturation of aseedling as compared to an untreated seed when applied in a seed soakapplication.

Example 12

An experiment was conducted to determine if the method of application ofa low concentration a mixotrophic Chlorella based composition to greenbean seeds (Phaseolus vulgaris) planted in soil affected the rate atwhich the seedlings emerge from the soil and mature. Green bean seedswere planted in trays with a potting soil mix of sphagnum moss, perlite,and vermiculite (2:1:1). Two treatments comprising a mixotrophicChlorella based composition were compared to an untreated control (UTC).The treatments were pasteurized, normalized to 10% solids, andstabilized with phosphoric acid (H₃PO₄) and potassium sorbate (C₆H₇KO₂),with the remaining balance consisting of water. The mixotrophicChlorella based composition was not previously frozen, and wasincorporated into the liquid composition for treatments used in thisexperiment directly after being harvested from the microalgae culturingsystem. The composition used in the treatments of this experiment wasnot analyzed to quantify bacteria in the composition, however aerobicplate counts for previous compositions prepared with the same componentsin the same manner contained 40,000-400,000 CFU/mL.

The mixotrophic Chlorella based liquid composition treatments wereapplied to the seeds at two different low concentrations, 4.7 mL/gallonor 8 mL/gallon, using the same treatment method. The testedconcentration of 4.7 mL/gallon diluted the composition which originallycontained 10% solids by weight of mixotrophic Chlorella whole cells tothe low percent solids content of only 0.012416%. The testedconcentration of 8 mL/gallon diluted the composition which originallycontained 10% solids by weight of mixotrophic Chlorella whole cells tothe low percent solids content of only 0.021134%. The treatment methodconsisted of drenching the soil from the top with 0.75 gallon of theliquid composition (equivalent to an application rate of 100gallons/acre) at the identified concentrations after planting the seeds.

Each of the two treatments were applied to two trays of 72 seeds. Visualobservations of the soil and plants were made daily to record how manyseeds had achieved emergence and maturation, as explained below. Thestandard used for assessing emergence was the achievement of thehypocotyl stage where a stem was visibly protruding from the pottingsoil mix. The standard used for assessing maturation was the achievementof the cotyledon stage where two leaves had visibly formed on theemerged stem. The experiment was conducted indoors with all seeds andtreatments subjected to the same controlled conditions includingtemperature, light, and supply of water. No other nutrients weresupplied during the experiment. Light supplied was artificial andprovided by fluorescent bulbs 24 hours a day. Results of the experimentare presented in Tables 44-49.

TABLE 44 Number of plants emerged by day 1 2 3 4 5 6 7 8 9 UntreatedControl — — 9 22 32 36 42 46 47 (UTC) 10% Mixotrophic — — 11 29 51 58 6263 64 Chlorella 4.7 mL 10% Mixotrophic — — 13 43 77 91 104 107 110Chlorella 8 mL

TABLE 45 % of total plants emerged by day 1 2 3 4 5 6 7 8 9 UntreatedControl 0 0 6 15 22 25 29 32 33 (UTC) 10% Mixotrophic 0 0 8 20 35 40 4344 44 Chlorella 4.7 mL 10% Mixotrophic 0 0 9 30 53 63 72 74 76 Chlorella8 mL

TABLE 46 % increase of plants emerged by day over the UTC 1 2 3 4 5 6 78 9 10% — — 22% 32%  59%  61%  48%  37%  36% Mixotrophic Chlorella 4.7mL 10% — — 44% 95% 141% 153% 148% 133% 134% Mixotrophic Chlorella 8 mL

As shown in the Tables 44-46, the 8 and 4.7 mL/gallon applicationsshowed consistently higher performance than the UTC regarding emergenceof the plants, with the 8 mL/gallon application consistently performingbetter than the 4.7 mL/gallon. The 4.7 mL/gallon application showed atleast a 22% and as much as a 61% increase over the UTC on comparativedays, and the 8 mL/gallon application demonstrated at least a 44% and asmuch as a 153% increase over the UTC. These results show that a lowconcentration of a mixotrophic Chlorella based composition is effectivein increasing the emergence of a seedling as compared to an untreatedseed when applied in a soil drench application.

TABLE 47 Number of plants matured by day 1 2 3 4 5 6 7 8 9 UntreatedControl — — 0 0 2 14 26 31 34 (UTC) 10% Mixotrophic — — 0 0 2 26 52 5758 Chlorella 4.7 mL 10% Mixotrophic — — 0 0 0 29 60 76 94 Chlorella 8 mL

TABLE 48 % of total plants matured by day 1 2 3 4 5 6 7 8 9 UntreatedControl 0 0 0 0 1 10 18 22 24 (UTC) 10% Mixotrophic 0 0 0 0 1 18 36 4040 Chlorella 4.7 mL 10% Mixotrophic 0 0 0 0 0 20 42 53 65 Chlorella 8 mL

TABLE 49 % increase of plants matured by day over the UTC 1 2 3 4 5 6 78 9 10% — — — —    0%  86% 100%  84%  71% Mixotrophic Chlorella 4.7 mL10% — — — — −100% 107% 131% 145% 176% Mixotrophic Chlorella 8 mL

As shown in the Tables 47-49, the 8 and 4.7 mL/gallon applicationsshowed consistently higher performance than the UTC regarding maturationof the plants, with the 8 mL/gallon application consistently performingbetter than the 4.7 mL/gallon. Starting on day 6, the 4.7 mL/gallonapplication showed at least a 71% and as much as a 100% increase overthe UTC on comparative days and the 8 mL/gallon application demonstratedat least a 107% and as much as a 176% increase over the UTC. Theincrease in maturation performance for the 8 mL/gallon application overthe UTC also increased over time. These results show that a lowconcentration of a mixotrophic Chlorella based composition is effectivein increasing the maturation of a seedling as compared to an untreatedseed when applied in a soil drench application.

With the characteristics that are shared among plants within theFabaceae plant family, the results shown in Examples 11-12 are likelyrepresentative as to the effectiveness of mixotrophic Chlorella basedcomposition as described throughout the specification on all plants inthe Fabaceae plant family, as well as on plants of other families.

Example 13

An experiment was conducted to determine if a low concentration and lowfrequency application of a mixotrophic Chlorella based composition tobell pepper plants by soil application affected the yield of the plants.Bell pepper (Capsicum annuum) are part of the Solanaceae plant familyand seeds were planted in a field in Ventura County, Calif. Twotreatments were compared to an untreated control (UTC) and are listed inTable 50. A commercially available macroalgae extract based product wasobtained from Acadian Seaplants Limited (30 Brown Avenue, Dartmouth,Nova Scotia, Canada, B3B 1X8) for comparison.

TABLE 50 Treatment No. Treatment Description 1 UTC - untreated watercheck 2 Mixotrophic Chlorella sp. - Whole Cells 3 Grower StandardProduct - Acadian Liquid Seaweed Concentrate

The mixotrophic Chlorella based composition was pasteurized, normalizedto 10% solids, and stabilized with phosphoric acid (H₃PO₄) and potassiumsorbate (C₆H₇KO₂), with the remaining balance consisting of water. Themixotrophic Chlorella whole cells were not previously subjected to apurification process to isolate the cells from the microalgae culturingmedium, nor were the cells previously subjected to a drying, extraction,or other process that can lyse or disrupt the cell walls. Themixotrophic Chlorella composition was previously frozen and thawed, andwas incorporated into the liquid composition for treatments used in thisexperiment after cold storage following being harvested from themicroalgae culturing system. The composition comprising mixotrophicChlorella used in the treatments of this experiment were not analyzed toquantify bacteria in the compositions, however aerobic plate counts forprevious compositions prepared with the same components in the samemanner contained 40,000-400,000 CFU/mL.

The mixotrophic Chlorella based composition was applied at a lowconcentration of 37.85 mL/gallon. The tested concentration of 37.85mL/gallon diluted the composition which originally contained 10% solidsby weight of mixotrophic Chlorella whole cells to the low percent solidscontent of only 0.099989%. The Acadian treatment was applied at aconcentration of 18.9 mL/gallon. Five total treatments were applied at alow frequency (i.e., averaging about 20 days between applications),starting three weeks after plant establishment. The treatments occurredwith 20 days between the first and second, 24 days between the secondand third, 11 days between the third and fourth, and 26 days between thefourth and fifth. The low concentration and low frequency treatmentswere applied by injection into a low volume irrigation drip systemsupplying water at a rate of 100 gallons/acre using a Hypro pumpoperating at 25 psi.

The experiment was set up as a block designed study of eight replicatesconsisting of 30 seeds each. Visual observations were used to evaluateplant vigor on a scale of 0-5, with 0 corresponding to plant death and 5corresponding to complete health. Production was evaluated by quality inthe two categories of marketable and unmarketable. Unmarketable fruitwas considered fruit which had heavy insect damage, blossom end rot,softness, and/or heavy sunburn. The field used in the experiment wasgrowing bell peppers for processing, and thus the quality needed forfresh market produce was not the target achievement. Additionally, thebell peppers were left in the field a length of time to ensure themaximum amount of reddening before harvest for processing. Thechlorophyll content was estimated by an SPAD value (Soil-Plant AnalysisDevelopment), a numeric value provided by a Minolta SPAD meter whichanalyzes the amount of light in a specific light spectrum passingthrough a leaf and converts that reading to a numerical value as anindicator of chlorophyll density in the leaf. Production was evaluatedby sampling based on picking all fruit to be found on two plants andreplicating this process eight times per treatment. All fruit wasweighed, counted, and reported as grams total weight per two plants andgrams total weight on average per fruit. All data rated as significantwas done so utilizing the Least Significant Difference analysis at a 90%confidence level, such that values with a statistical significantidentifier of the same letter are not significantly different. Resultsare shown in Tables 51-65 for the treatments designated with an S forsoil application, along with accompanying statistical significanceidentifiers.

Example 14

An experiment was conducted to determine if a low concentration and lowfrequency application of mixotrophic Chlorella based composition to bellpepper plants (Capsicum annuum) by foliar application affected the yieldof the plants. The foliar trial occurred in the same location, with thesame treatments, and with the same design as the experiment of Example13.

The mixotrophic Chlorella based composition was applied at a lowconcentration of 7 mL/gallon. The tested concentration of 7 mL/gallondiluted the composition which originally contained 10% solids by weightof mixotrophic Chlorella whole cells to the low percent solids contentof only 0.018492%. The Acadian treatment was applied at a concentrationof 18.9 mL/gallon. Five total treatments were applied at a low frequency(i.e., averaging about 21 days between applications), starting threeweeks after plant establishment. The treatments occurred with 20 daysbetween the first and second, 23 days between the second and third, 15days between the third and fourth, and 27 days between the fourth andfifth. The low concentration and low frequency treatments were applieddirectly to the foliage at a rate of 25 gallons/acre with a backpacksprayer operating at 40 psi through a Hollow Co. nozzle size D-6.

All data rated as significant was done so utilizing the LeastSignificant Difference analysis at a 90% confidence level, such thatvalues with a statistical significant identifier of the same letter arenot significantly different. Results are shown in Tables 51-65 for thetreatments designated with an F for foliar application, along withaccompanying statistical significance identifiers. It was noted by thetime the field was harvested many of the above mentioned unmarketablequality issues did occur and thus the ratio of unmarketable fruit washigher in this field than one might expect.

TABLE 51 Plant Sizing - Whole plant (grams) (A = early, B = later)Increase Increase Avg. A over UTC Avg. B over UTC 1 UTC - untreatedwater check F 4.3 a 31.2 a UTC - untreated water check S 4.4 a 24.8 2Mixotrophic Chlorella sp. - 4.6 a 6% 30.9 a −1% Whole Cells FMixotrophic Chlorella sp. - 4.4 a −1%  26.7 a  8% Whole Cells S 3 GrowerStandard Product - 4.5 a 4% 35.6 a 14% Acadian Liquid SeaweedConcentrate F Grower Standard Product - 5.1 a 17%  32.7 a 32% AcadianLiquid Seaweed Concentrate S

Table 51 shows that there was not statistical significance to theresults of the mixotrophic Chlorella based composition treatmentscompared to the UTC regarding whole plant weight. The foliar applicationof mixotrophic Chlorella based composition performed better than thesoil application at the first measurement and resulted in a 6% increaseover the UTC but did not sustain the advantage at the secondmeasurement. The soil application performed better at the secondmeasurement than the foliar application and resulted in an 8% increaseover the UTC.

TABLE 52 Plant Sizing - Root (grams) (A = earlier, B = later) IncreaseIncrease Avg. A over UTC Avg. B over UTC 1 UTC - untreated water check F0.6 a 3.4 a UTC - untreated water check S 0.6 a 3.0 2 MixotrophicChlorella sp. - 0.6 a 7% 3.3 a −4% Whole Cells F Mixotrophic Chlorellasp. - 0.7 a 8% 3.3 a  9% Whole Cells S 3 Grower Standard Product - 0.6 a0% 4.0 a 17% Acadian Liquid Seaweed Concentrate F Grower StandardProduct - 0.7 a 8% 3.6 a 21% Acadian Liquid Seaweed Concentrate S

Table 52 shows that there was not statistical significance to theresults of the mixotrophic Chlorella based composition treatmentscompared to the UTC regarding root weight. The foliar and soilapplications of mixotrophic Chlorella based composition performed betterthan the UTC at the first measurement, with 7% and 8% increases over theUTC. The foliar application did not sustain this advantage at the secondmeasurement, but the soil application maintained the advantage showing a9% increase over the UTC.

TABLE 53 Plant Sizing - Shoot (grams) (A = earlier, B = later) IncreaseIncrease Avg. A over UTC Avg. B over UTC 1 UTC - untreated water check F3.8 a 27.7 a UTC - untreated water check S 3.7 a 24.0 2 MixotrophicChlorella sp. - 4.0 a 6% 27.6 a  0% Whole Cells F Mixotrophic Chlorellasp. - 3.7 a −2%  23.5 a −2% Whole Cells S 3 Grower Standard Product -3.9 a 5% 31.6 a 14% Acadian Liquid Seaweed Concentrate F Grower StandardProduct - 4.4 a 18%  29.1 a 21% Acadian Liquid Seaweed Concentrate S

Table 53 shows that there was not statistical significance to theresults of the mixotrophic Chlorella based composition treatmentscompared to the UTC regarding shoot weight. The foliar application ofmixotrophic Chlorella based composition performed better than the UTCand soil application at the first measurement, with a 6% increases overthe UTC. The foliar application did not sustain this advantage at thesecond measurement.

TABLE 54 Average Plant Chlorophyll Content (SPAD) Increase over A B Avg.UTC 1 UTC - untreated water 64.7 — 39.7 a 52.2 check F UTC - untreatedwater — 69.7 ab 69.7 check S 2 Mixotrophic Chlorella sp. - 71.5 — 36.4 a54.0 3% Whole Cells F Mixotrophic Chlorella sp. - — 70.2 ab 70.2 1%Whole Cells S 3 Grower Standard Product - 70.6 — 35.4 a 53.0 2% AcadianLiquid Seaweed Concentrate F Grower Standard Product - — 64.5 a 64.5−7%  Acadian Liquid Seaweed Concentrate S

Table 54 shows that there was not statistical significance to theresults of the mixotrophic Chlorella based composition treatmentscompared to the UTC regarding chlorophyll content. The foliar and soilapplications of mixotrophic Chlorella based composition performed within3% of the UTC.

TABLE 55 Average Plant Vigor (Visual Scale 0-5) Increase over A B C Avg.UTC 1 UTC - untreated water 3.4 a 4.5 a 4.0 a 4.0 check F UTC -untreated water 3.5 a 4.5 a 4.0 check S 2 Mixotrophic Chlorella 3.2 a4.1 a 4.0 a 3.8 −5% sp. - Whole Cells F Mixotrophic Chlorella 4.0 a 4.0a 4.0  0% sp. - Whole Cells S 3 Grower Standard 3.2 a 4.3 a 4.0 a 3.8−3% Product - Acadian Liquid Seaweed Concentrate F Grower Standard 3.5 a4.0 a 3.8 −6% Product - Acadian Liquid Seaweed Concentrate S

Table 55 shows that there was not statistical significance to theresults of the mixotrophic Chlorella based composition treatmentscompared to the UTC regarding plant vigor, nor was there a numericaladvantage.

TABLE 56 Total Unmarketable Plant Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 1895.0 a UTC - untreated watercheck S 963.8 a 2 Mixotrophic Chlorella sp. - 1803.1 a  −5% Whole CellsF Mixotrophic Chlorella sp. - 179.4 b −81% Whole Cells S 3 GrowerStandard Product - 1580.6 a −17% Acadian Liquid Seaweed Concentrate FGrower Standard Product - 66.9 b −93% Acadian Liquid Seaweed ConcentrateS

Table 56 shows that the soil application of the mixotrophic Chlorellabased composition had a statistically significant decrease inunmarketable plant weight compared to the UTC, and the foliarapplication results were not statistically significant compared to theUTC.

TABLE 57 Total Unmarketable Plant Yield per Plot (number) Increase Avg.over UTC 1 UTC - untreated water check F 10.8 a UTC - untreated watercheck S 6.0 a 2 Mixotrophic Chlorella sp. - 9.8 a  −9% Whole Cells FMixotrophic Chlorella sp. - 1.9 b −69% Whole Cells S 3 Grower StandardProduct - 9.1 a −15% Acadian Liquid Seaweed Concentrate F GrowerStandard Product - 1.1 b −81% Acadian Liquid Seaweed Concentrate S

Table 57 shows that the soil application of the mixotrophic Chlorellabased composition had a statistically significant decrease inunmarketable plant yield compared to the UTC, and the foliar applicationresults were not statistically significant compared to the UTC.

TABLE 58 Total Unmarketable Fruit Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 178.5 a UTC - untreated watercheck S 56.2 a 2 Mixotrophic Chlorella sp. - 182.8 a 2% Whole Cells FMixotrophic Chlorella sp. - 57.6 a 2% Whole Cells S 3 Grower StandardProduct - 173.2 a −3%  Acadian Liquid Seaweed Concentrate F GrowerStandard Product - 35.3 a −37%  Acadian Liquid Seaweed Concentrate S

Table 58 shows that the soil and foliar applications of the mixotrophicChlorella based composition were not statistically significant comparedto the UTC for unmarketable fruit weight, but both showed a numericalincrease of 2% over the UTC. The soil application of mixotrophicChlorella based composition also outperformed the Acadian product, whichshowed a 37% decrease compared to the UTC.

TABLE 59 Total Marketable Plant Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 120.6 a UTC - untreated watercheck S 317.5 c 2 Mixotrophic Chlorella sp. - 386.3 a 220% Whole Cells FMixotrophic Chlorella sp. - 1224.4 a 286% Whole Cells S 3 GrowerStandard Product - 502.5 a 317% Acadian Liquid Seaweed Concentrate FGrower Standard Product - 1233.1 a 288% Acadian Liquid SeaweedConcentrate S

Table 59 shows that the results of the soil application of themixotrophic Chlorella based composition were statistically significantcompared to the UTC for marketable plant weight, and both soil andfoliar applications showed large numerical increases of 286% and 220%over the UTC, which was comparable with the commercially successfulAcadian product. These results show that small amounts of themixotrophic Chlorella based composition at a low concentration and lowfrequency application are effective for not only improving plant weight,put improving plant weight in the higher quality plants (i.e.,marketable) when applied to the soil or foliage.

TABLE 60 Total Marketable Plant Yield per Plot (number) Increase Avg.over UTC 1 UTC - untreated water check F 0.6 a UTC - untreated watercheck S 2.3 a 2 Mixotrophic Chlorella sp. - 2.0 a 220% Whole Cells FMixotrophic Chlorella sp. - 6.8 a 200% Whole Cells S 3 Grower StandardProduct - 2.8 a 340% Acadian Liquid Seaweed Concentrate F GrowerStandard Product - 7.1 a 217% Acadian Liquid Seaweed Concentrate S

Table 60 shows that the results of the soil and foliar applications ofthe mixotrophic Chlorella based composition showed large numericalincreases of 200% and 220% over the UTC, which was comparable with thecommercially successful Acadian product. These results show that smallamounts of the mixotrophic Chlorella based composition at a lowconcentration and low frequency application are effective for not onlyimproving plant yield, put improving plant yield in the higher qualityplants (i.e., marketable) when applied to the soil or foliage.

TABLE 61 Total Marketable Fruit Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 73.1 a UTC - untreated watercheck S 123.7 b 2 Mixotrophic Chlorella sp. - 43.8 a −40% Whole Cells FMixotrophic Chlorella sp. - 182.9 a 48% Whole Cells S 3 Grower StandardProduct - 115.8 a 58% Acadian Liquid Seaweed Concentrate F GrowerStandard Product - 66.9 a −46% Acadian Liquid Seaweed Concentrate S

Table 61 shows that the results of the soil application of themixotrophic Chlorella based composition were statistically significantcompared to the UTC for marketable fruit weight. The soil application ofmixotrophic Chlorella based composition also showed a numerical increaseof 48% over the UTC. The soil application of Chlorella based compositionalso outperformed the Acadian product, which showed a 46% decreasecompared to the UTC. These results show that small amounts of themixotrophic Chlorella based composition at a low concentration and lowfrequency application are effective for not only improving fruit weight,but improving fruit weight in the higher quality plants (i.e.,marketable) when applied to the soil.

TABLE 62 Total Production Plant Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 2015.6 a UTC - untreated watercheck S 656.3 c 2 Mixotrophic Chlorella sp. - 2189.4 a 9% Whole Cells FMixotrophic Chlorella sp. - 1403.8 a 114% Whole Cells S 3 GrowerStandard Product - 2083.1 a 3% Acadian Liquid Seaweed Concentrate FGrower Standard Product - 1300.0 a 98% Acadian Liquid SeaweedConcentrate S

Table 62 shows that the results of the soil application of themixotrophic Chlorella based composition were statistically significantcompared to the UTC for production plant weight. The soil application ofmixotrophic Chlorella based composition also showed a numerical increaseof 114% over the UTC, with the foliar application showing a 9% increaseover the UTC, which were both comparable to the Acadian product. Theseresults show that small amounts of the mixotrophic Chlorella basedcomposition at a low concentration and low frequency application areeffective for not only total production plant weight when applied to thesoil.

TABLE 63 Total Production Plant Yield per Plot (number) Increase Avg.over UTC 1 UTC - untreated water check F 11.4 a UTC - untreated watercheck S 8.3 a 2 Mixotrophic Chlorella sp. - 11.8 a 3% Whole Cells FMixotrophic Chlorella sp. - 8.6 a 5% Whole Cells S 3 Grower StandardProduct - 11.9 a 4% Acadian Liquid Seaweed Concentrate F Grower StandardProduct - 8.3 a 0% Acadian Liquid Seaweed Concentrate S

Table 63 shows that the results of the soil and foliar applications ofthe mixotrophic Chlorella based composition were not statisticallysignificant compared to the UTC for production plant yield, but did shownumerical increases of 5% and 3% over the UTC.

TABLE 64 Average Production Fruit Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 179.0 a UTC - untreated watercheck S 80.5 b 2 Mixotrophic Chlorella sp. - 189.6 a 6% Whole Cells FMixotrophic Chlorella sp. - 167.0 a 107% Whole Cells S 3 Grower StandardProduct - 174.1 a −3% Acadian Liquid Seaweed Concentrate F GrowerStandard Product - 159.8 a 98% Acadian Liquid Seaweed Concentrate S

Table 64 shows that the results of the soil application of themixotrophic Chlorella based composition were statistically significantcompared to the UTC for production fruit weight. The soil application ofmixotrophic Chlorella based composition also showed a numerical increaseof 117% over the UTC, with the foliar application showing a 6% increaseover the UTC, both of which were comparable to the Acadian product.These results show that small amounts of the mixotrophic Chlorella basedcomposition at a low concentration and low frequency application areeffective for not only total production fruit weight when applied to thesoil or foliage.

TABLE 65 Utilization (%, ratio of marketable fruit to total fruitproduced by weight) Increase Avg. over UTC 1 UTC - untreated water checkF 6.5 a UTC - untreated water check S 45.0 b 2 Mixotrophic Chlorellasp. - 11.8 a 81% Whole Cells F Mixotrophic Chlorella sp. - 88.3 a 96%Whole Cells S 3 Grower Standard Product - 18.3 a 181% Acadian LiquidSeaweed Concentrate F Grower Standard Product - 94.6 a 110% AcadianLiquid Seaweed Concentrate S

Table 65 shows that the results of the soil application of themixotrophic Chlorella based composition were statistically significantcompared to the UTC for utilization percentage (ratio of marketablefruit to total fruit produced by weight). The soil application ofmixotrophic Chlorella based composition also showed a numerical increaseof 96% over the UTC, with the foliar application showing an 81% increaseover the UTC. These results show that small amounts of the mixotrophicChlorella based composition at a low concentration and low frequencyapplication are effective for improving the total quality of the fieldwhen applied to the soil or foliage.

Example 15

An experiment was conducted to determine if a low concentration and lowfrequency application of a mixotrophic Chlorella based composition togavilon tomato plants (Solanum lycopersicum) by soil applicationaffected the yield of the plants. Tomatoes are also members of theSolanaceae plant family. The soil application trial occurred in the samelocation, with the same treatments, and with the same design as theexperiment of Example 13. The tomato plants were grown as a bush on theground for this experiment.

The mixotrophic Chlorella based composition was applied at a lowconcentration of 37.85 mL/gallon. The tested concentration of 37.85mL/gallon diluted the composition which originally contained 10% solidsby weight of mixotrophic Chlorella whole cells to the low percent solidscontent of only 0.099989%. The Acadian treatment was applied at aconcentration of 18.9 mL/gallon. Five total treatments were applied at alow frequency (i.e., averaging about 23 days between applications),starting three weeks after plant establishment. The treatments occurredwith 19 days between the first and second, 29 days between the secondand third, 23 days between the third and fourth, and 21 days between thefourth and fifth. The low concentration and low frequency treatmentswere applied by injection into a low volume irrigation drip system at arate of 100 gallons/acre using a Hypro pump operating at 25 psi.

All data rated as significant was done so utilizing the LeastSignificant Difference analysis at a 90% confidence level, such thatvalues with a statistical significant identifier of the same letter arenot significantly different. Results are shown in tables 66-78 for thetreatments designated with an S for soil application, along withaccompanying statistical significance identifiers.

Example 16

An experiment was conducted to determine if a low concentration and lowfrequency application of mixotrophic Chlorella based composition togavilon tomato plants (Solanum lycopersicum) by foliar applicationaffected the yield of the plants. The foliar trial occurred in the samelocation, with the same treatments, and with the same design as theexperiment of Example 14. The tomato plants were grown on stakes forthis experiment.

The mixotrophic Chlorella based composition was applied at a lowconcentration of 7 mL/gallon. The tested concentration of 7 mL/gallondiluted the composition which originally contained 10% solids by weightof mixotrophic Chlorella whole cells to the low percent solids contentof only 0.018492%. The Acadian treatment was applied at a concentrationof 18.9 mL/gallon. Five total treatments were applied at a low frequency(i.e., averaging about 21 days between applications), starting threeweeks after plant establishment. The treatments occurred with 19 daysbetween the first and second, 21 days between the second and third, 23days between the third and fourth, and 21 days between the fourth andfifth. The low concentration and low frequency treatments were applieddirectly to the foliage at a rate of 25 gallons/acre with a backpacksprayer operating at 40 psi through a Hollow Co. nozzle size D-6.

All data rated as significant was done so utilizing the LeastSignificant Difference analysis at a 90% confidence level, such thatvalues with a statistical significant identifier of the same letter arenot significantly different. Results are shown in tables 66-78 for thetreatments designated with an F for foliar application, along withaccompanying statistical significance identifiers.

TABLE 66 Average Plant Chlorophyll Content (SPAD) Increase over A B Avg.UTC 1 UTC - untreated water 52.7 a 48.0 a 50.4 check F UTC - untreatedwater 44.6 a 44.6 check S 2 Mixotrophic Chlorella sp. - 54.6 a 45.4 a50.0 −1% Whole Cells F Mixotrophic Chlorella sp. - 44.5 a 44.5  0% WholeCells S 3 Grower Standard Product - 53.9 a 46.2 a 50.1 −1% AcadianLiquid Seaweed Concentrate F Grower Standard Product - 41.8 a 41.8 −6%Acadian Liquid Seaweed Concentrate S

Table 66 shows that there was not statistical significance to theresults of the mixotrophic Chlorella based composition treatmentscompared to the UTC regarding chlorophyll content, nor a numericalincrease.

TABLE 67 Average Plant Vigor (Visual Scale 0-5) Increase over A B Avg.UTC 1 UTC - untreated water check F 4.9 a 3.9 a 4.4 UTC - untreatedwater check S 4.2 a 4.2 2 Mixotrophic Chlorella sp. - 5.0 a 3.6 a 4.3−2% Whole Cells F Mixotrophic Chlorella sp. - 4.5 a 4.5  7% Whole CellsS 3 Grower Standard Product - 4.9 a 4.1 a 4.5  2% Acadian Liquid SeaweedConcentrate F Grower Standard Product - 4.1 a 4.1 −2% Acadian LiquidSeaweed Concentrate S

Table 67 shows that there was not statistical significance to theresults of the mixotrophic Chlorella based composition treatmentscompared to the UTC regarding plant vigor, however the soil applicationshowed a 7% increase over the UTC.

TABLE 68 Total Unmarketable Plant Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 205.8 a UTC - untreated watercheck S 2156.0 a 2 Mixotrophic Chlorella sp. - 139.2 a −32% Whole CellsF Mixotrophic Chlorella sp. - 2279.2 a 6% Whole Cells S 3 GrowerStandard Product - 162.5 a −21% Acadian Liquid Seaweed Concentrate FGrower Standard Product - 997.5 b −54% Acadian Liquid SeaweedConcentrate S

Table 68 shows that the application of the mixotrophic Chlorella basedcomposition did not have a statistically significant decrease inunmarketable plant weight compared to the UTC, however the foliarapplication showed a 32% decrease over the UTC. The soil application ofmixotrophic Chlorella based composition showed a 6% increase over theUTC, while the commercially successful Acadian product soil applicationshowed a 54% decrease.

TABLE 69 Total Unmarketable Plant Yield per Plot (number) Increase Avg.over UTC 1 UTC - untreated water check F 5.8 a UTC - untreated watercheck S 49.3 a 2 Mixotrophic Chlorella sp. - 3.0 a −49% Whole Cells FMixotrophic Chlorella sp. - 47.7 a −3% Whole Cells S 3 Grower StandardProduct - 3.0 a −49% Acadian Liquid Seaweed Concentrate F GrowerStandard Product - 28.5 bc −42% Acadian Liquid Seaweed Concentrate S

Table 69 shows that the application of the mixotrophic Chlorella basedcomposition did not have a statistically significant decrease inunmarketable plant yield compared to the UTC, however the foliarapplication showed a 49% decrease and the soil application showed a 3%decrease with respect to the UTC, which was smaller than the 42%decrease of the Acadian product soil application.

TABLE 70 Total Unmarketable Fruit Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 29.6 a UTC - untreated watercheck S 45.8 a 2 Mixotrophic Chlorella sp. - 27.5 a −7% Whole Cells FMixotrophic Chlorella sp. - 47.4 a 3% Whole Cells S 3 Grower StandardProduct - 35.5 a 20% Acadian Liquid Seaweed Concentrate F GrowerStandard Product - 34.8 a −24% Acadian Liquid Seaweed Concentrate S

Table 70 shows that the soil and foliar applications of the mixotrophicChlorella based composition were not statistically significant comparedto the UTC for unmarketable fruit weight, but the soil applicationshowed a 3% increase, while the Acadian product showed a 24% decrease,and the foliar application showed a 7% decrease with respect to the UTC.

TABLE 71 Total Marketable Plant Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 8702.5 a UTC - untreated watercheck S 7616.7 a 2 Mixotrophic Chlorella sp. - 8317.5 a −4% Whole CellsF Mixotrophic Chlorella sp. - 8160.8 a 7% Whole Cells S 3 GrowerStandard Product - 7731.7 a −11% Acadian Liquid Seaweed Concentrate FGrower Standard Product - 7828.3 a 3% Acadian Liquid Seaweed ConcentrateS

Table 71 shows that the results of the application of the mixotrophicChlorella based composition were not statistically significant comparedto the UTC for marketable plant weight, however the soil applicationshowed a 7% increase over the UTC.

TABLE 72 Total Marketable Plant Yield per Plot (number) Increase Avg.over UTC 1 UTC - untreated water check F 120.8 a UTC - untreated watercheck S 103.5 a 2 Mixotrophic Chlorella sp. - 103.0 a −15% Whole Cells FMixotrophic Chlorella sp. - 115.3 a 11% Whole Cells S 3 Grower StandardProduct - 107.7 a −11% Acadian Liquid Seaweed Concentrate F GrowerStandard Product - 118.0 a 14% Acadian Liquid Seaweed Concentrate S

Table 72 shows that the results of the soil and foliar applications ofthe mixotrophic Chlorella based composition were not statisticallysignificant compared to the UTC for marketable plant yield, however thesoil application showed an 11% increase over the UTC.

TABLE 73 Total Marketable Fruit Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 72.2 b UTC - untreated watercheck S 74.5 a 2 Mixotrophic Chlorella sp. - 80.5 a 11% Whole Cells FMixotrophic Chlorella sp. - 70.1 a −6% Whole Cells S 3 Grower StandardProduct - 72.0 b 0% Acadian Liquid Seaweed Concentrate F Grower StandardProduct - 65.7 a −12% Acadian Liquid Seaweed Concentrate S

Table 73 shows that the results of the foliar application of themixotrophic Chlorella based composition were statistically significantcompared to the UTC and Acadian product for marketable fruit weight, andresulted in an 11% increase over the UTC. These results show that smallamounts of the mixotrophic Chlorella based composition at a lowconcentration and low frequency application are effective for not onlyimproving fruit weight, put improving fruit weight in the higher qualityplants (i.e., marketable) when applied to the foliage.

TABLE 74 Total Production Plant Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 8908.3 a UTC - untreated watercheck S 9272.7 a 2 Mixotrophic Chlorella sp. - 8456.7 a −5% Whole CellsF Mixotrophic Chlorella sp. - 10440.0 a 13% Whole Cells S 3 GrowerStandard Product - 7894.2 a −11% Acadian Liquid Seaweed Concentrate FGrower Standard Product - 8825.8 a −5% Acadian Liquid SeaweedConcentrate S

Table 74 shows that the results of the soil application of themixotrophic Chlorella based composition were not statisticallysignificant compared to the UTC for production plant weight, however thesoil application resulted in a numerical increase of 13% over the UTCwhile the Acadian product showed a 5% decrease.

TABLE 75 Total Production Plant Yield per Plot (number) Increase Avg.over UTC 1 UTC - untreated water check F 126.7 a UTC - untreated watercheck S 152.8 ab 2 Mixotrophic Chlorella sp. - 110.2 a −13% Whole CellsF Mixotrophic Chlorella sp. - 163.0 a 7% Whole Cells S 3 Grower StandardProduct - 110.7 a −13% Acadian Liquid Seaweed Concentrate F GrowerStandard Product - 146.5 abc −4% Acadian Liquid Seaweed Concentrate S

Table 75 shows that the results of the soil and foliar applications ofthe mixotrophic Chlorella based composition were not statisticallysignificant compared to the UTC for production plant yield, but the soilapplication showed an increase of 7% over the UTC, with the Acadianproduct showing a 4% decrease.

TABLE 76 Average Production Fruit Weight per Plot (grams) Increase Avg.over UTC 1 UTC - untreated water check F 70.7 a UTC - untreated watercheck S 64.8 a 2 Mixotrophic Chlorella sp. - 76.7 a 9% Whole Cells FMixotrophic Chlorella sp. - 63.1 a −3% Whole Cells S 3 Grower StandardProduct - 71.6 a 1% Acadian Liquid Seaweed Concentrate F Grower StandardProduct - 59.4 a −8% Acadian Liquid Seaweed Concentrate S

Table 76 shows that the results of the foliar application of themixotrophic Chlorella based composition were not statisticallysignificant compared to the UTC for production fruit weight, however thefoliar application showed a numerical increase of 9% over the UTC.

TABLE 77 Utilization (%, the ratio of marketable fruit to total fruitproduced by weight) Increase Avg. over UTC 1 UTC - untreated water checkF 97.5 a UTC - untreated water check S 76.8 c 2 Mixotrophic Chlorellasp. - 98.3 a 1% Whole Cells F Mixotrophic Chlorella sp. - 77.2 c 0%Whole Cells S 3 Grower Standard Product - 98.0 a 1% Acadian LiquidSeaweed Concentrate F Grower Standard Product - 88.7 a 15% AcadianLiquid Seaweed Concentrate S

Table 77 shows that the results of the application of the mixotrophicChlorella based composition were not statistically significant comparedto the UTC for utilization percentage (ratio of marketable fruit tototal fruit produced by weight).

With the characteristics that are shared among plants within theSolanaceae plant family, the results shown in the Examples 6-10 and13-16, are likely representative as to the effectiveness of amixotrophic Chlorella based composition as described by throughout thespecification on all plants in the Solanaceae plant family, as well asplants in other families.

Example 17

An experiment was conducted to determine the effects of differentapplication rates of a low concentration mixotrophic Chlorella basedcomposition (designated as “PT” or as “PhycoTerra” in various Figurelegends throughout this disclosure) on plants. Under hydroponicconditions, the composition was applied between 3 mL/gal-150 mL/gal. Anutrient only mock composition was also tested. The mock compositioncontained only non-biological components. All treatment conditionsincluded fertilizer. The control was a fertilizer only treatment (Vegonly). The experiment demonstrated that the composition has biologicaleffects on plants at low concentrations. The results of the experimentare shown in FIG. 4.

Example 18

Experiments were conducted to determine if application of a lowconcentration mixotrophic Chlorella based composition to plants thatwere salt-stressed at time of seeding affected the yield of the plants.Results of such experiments are shown in FIG. 5A-5F. In one experiment,pole beans were soaked in city water or 18 mL/gallon of themicroalgae-based composition for four hours. The seeds were planted incoco (an inert coconut fiber medium) and grown in a hydroponicsplatform. Seeds were planted (without washing) in a randomized fashionand treated with 80 mMol salt the first day. To test the importance ofrun-off (RO), half of each seed soaking type was either watered with 57mL of RO water (full saturation), or 171 mL of 80 mMol salt solution(Heavy run-off). Some cells contained just the coco and were harvestedto look at salinity profile in the coco with and without run off.Effects of the treatments were assessed by measuring dry weight,circumference, and photosynthetic yield (Y[ii]) (FIG. 5A). Total freshweight (FIG. 5B) and shoot weight (FIG. 5C) were also assessed. Theresults also show that seeds soaked in the mixotrophic Chlorella basedcomposition before planting had a 40% increase in germination rate (FIG.5D). In addition, seeds soaked in the mixotrophic Chlorella basedcomposition germinated earlier than seeds soaked in City Water, andseeds watered with RO water to full saturation had a decrease ingermination compared to seeds watered with 80 mMol salt solution withheavy run-off (FIG. 5E).

Example 19

An experiment was conducted to determine if an application of a lowconcentration mixotrophic Chlorella based composition (PT) to plantsthat were drought stressed at time of seeding affected the yield of theplants. The conditions were (1) controls: outside heat but saturated,(2) no water+outside heat, and (3) no water+outside heat+1 hr sun.Results of the experiment show that plants treated with PT showed nodetrimental effects when exposed to drought and sun whereas plants withno PT treatment reacted to drought and sun.

Example 20

Experiments were conducted to determine if an application of a lowconcentration mixotrophic Chlorella based composition (designated“PhycoTerra” or “PT” in figure legends) to turf affected the yield ofthe plants. In the experiments, the composition was applied to turfgrassat 6 different concentrations with urea. The concentrations ranged from0.3-15 L/acre and the turf was treated at either 14 or 21 day intervals.These treatments were compared to turf samples that were given (1) notreatment (UTC), (2) Acadian (a seaweed extract composition) and (2)urea only. The results of the experiment are shown in FIG. 6A-6B. Theshoot weight was shown to respond significantly to the treatment: At anapplication rate of 15 L/acre with an interval of 14 days, the shootweight was found to be <30% higher than the untreated control (UTC) andall application rates gave significantly higher shoot weight than wasachieved with application of Acadian at a 21-day interval ofapplication.

Example 21

Experiments were conducted to determine if application of a lowconcentration mixotrophic Chlorella based composition (PT) to peanutsaffected the yield of the plants. In the experiments, the compositionwas applied at 5 different concentrations. These treatments werecompared to plants that were given no treatment (UTC) and to plants thatwere treated with Acadian (a seaweed extract composition). In theexperiments, the soil around in the plants was tested for pH and humicacid. As known in the art, a pH soil range of 5.5-6.5 is ideal forpeanuts. Humic acid is an indicator of decomposition in the soil. Theexperiments show that the plot weight was significantly increased withPT given at 75.7 mL/gal-300 mL/gal and that the seed weight likewise wassignificantly increased with PT given at 300 mL/gal. The results of theexperiment are shown in the Table 78 below and FIG. 7A-7B.

TABLE 78 Level Least Sq Mean PT (150 mL/gal) A 8743.0288 PT (75.7mL/gal) A 8555.8791 PT (300 mL/gal) A 8181.6657 Acadian (18.9 mL/gal) AB 7739.4135 PT (37.8 mL/gal) A B 7626.0155 PT (18.9 mL/gal) A B7268.8118 UTC B 6367.2977 PT (300 mL/gal) A 1259.7500 Acadian (18.9mL/gal) A B 1167.2500 PT (150 mL/gal) A B 1127.0000 PT (75.7 mL/gal) A B1112.2500 PT (37.8 mL/gal) A B 1096.5000 PT (18.9 mL/gal) A B 1028.5000UTC B 916.2500

Example 22

Experiments were conducted to determine if an application of a lowconcentration mixotrophic Chlorella based composition to Basil plantsaffected the yield of the plants. In the experiments, the compositionwas applied after/in addition to an application of a commercialhydroponics fertilizer. This treatment was compared with fertilizeralone as a control. In these experiments, plants exposed to thetreatment, and other plants exposed to the control, were compared basedupon measurements of stem diameter and plant fresh weight. While thestem diameter showed no significant difference between the treatment andthe control, the measure of fresh weight did demonstrate that thecomposition has a positive effect on plant growth, as compared with thefertilizer alone.

Example 23—Fabaceae (Leguminosae)

Experiments are conducted to test effects of application of a microalgaebased composition to crop plants of the family Fabaceae (Leguminosae).Application is done as in other examples herein, such that, in varioustreatments, (a) seeds are wetted or soaked in the composition; (b) thecomposition is applied to soil pre-germination; (c) the composition isapplied to soil post-germination; (d) the composition is appliedperiodically to soil during the growing season; and/or (e) thecomposition is applied to leaves of the plants once or periodicallyduring the growing season. Results are measures for appropriate plantcharacteristics including: seed germination rate, seed germination time,seedling emergence, seedling emergence time, seedling size, plant freshweight, plant dry weight, utilization, fruit production, leafproduction, leaf formation, thatch height, plant health, plantresistance to salt stress, plant resistance to heat stress, plantresistance to heavy-metal stress, plant resistance to drought,maturation time, yield, root length, root mass, color, insect damage,blossom end rot, softness, fruit quality, and sunburn. Results show atleast a 10% quantitative improvement as to at least one characteristicunder at least one mode of application (a-e) of the composition. In someembodiments, results show at least a 25% quantitative improvement in atleast one characteristic and/or a statistically significant improvementin at least two characteristics.

Example 24—Poaceae

Experiments are conducted to test effects of application of a microalgaebased composition to crop plants of the family Poaceae. Application isdone as in other examples herein, such that, in various treatments, (a)seeds are wetted or soaked in the composition; (b) the composition isapplied to soil pre-germination; (c) the composition is applied to soilpost-germination; (d) the composition is applied periodically to soilduring the growing season; and/or (e) the composition is applied toleaves of the plants once or periodically during the growing season.Results are measures for appropriate plant characteristics including:seed germination rate, seed germination time, seedling emergence,seedling emergence time, seedling size, plant fresh weight, plant dryweight, utilization, fruit production, leaf production, leaf formation,thatch height, plant health, plant resistance to salt stress, plantresistance to heat stress, plant resistance to heavy-metal stress, plantresistance to drought, maturation time, yield, root length, root mass,color, insect damage, blossom end rot, softness, fruit quality, andsunburn. Results show at least a 10% quantitative improvement as to atleast one characteristic under at least one mode of application (a-e) ofthe composition. In some embodiments, results show at least a 25%quantitative improvement in at least one characteristic and/or astatistically significant improvement in at least two characteristics.

Example 25—Roasaceae

Experiments are conducted to test effects of application of a microalgaebased composition to crop plants of the family Roasaceae. Application isdone as in other examples herein, such that, in various treatments, (a)seeds are wetted or soaked in the composition; (b) the composition isapplied to soil pre-germination; (c) the composition is applied to soilpost-germination; (d) the composition is applied periodically to soilduring the growing season; and/or (e) the composition is applied toleaves of the plants once or periodically during the growing season.Results are measures for appropriate plant characteristics including:seed germination rate, seed germination time, seedling emergence,seedling emergence time, seedling size, plant fresh weight, plant dryweight, utilization, fruit production, leaf production, leaf formation,thatch height, plant health, plant resistance to salt stress, plantresistance to heat stress, plant resistance to heavy-metal stress, plantresistance to drought, maturation time, yield, root length, root mass,color, insect damage, blossom end rot, softness, fruit quality, andsunburn. Results show at least a 10% quantitative improvement as to atleast one characteristic under at least one mode of application (a-e) ofthe composition. In some embodiments, results show at least a 25%quantitative improvement in at least one characteristic and/or astatistically significant improvement in at least two characteristics.

Example 26—Vitaceae

Experiments are conducted to test effects of application of a microalgaebased composition to crop plants of the family Vitaceae. Application isdone as in other examples herein, such that, in various treatments, (a)seeds are wetted or soaked in the composition; (b) the composition isapplied to soil pre-germination; (c) the composition is applied to soilpost-germination; (d) the composition is applied periodically to soilduring the growing season; and/or (e) the composition is applied toleaves of the plants once or periodically during the growing season.Results are measures for appropriate plant characteristics including:seed germination rate, seed germination time, seedling emergence,seedling emergence time, seedling size, plant fresh weight, plant dryweight, utilization, fruit production, leaf production, leaf formation,thatch height, plant health, plant resistance to salt stress, plantresistance to heat stress, plant resistance to heavy-metal stress, plantresistance to drought, maturation time, yield, root length, root mass,color, insect damage, blossom end rot, softness, fruit quality, andsunburn. Results show at least a 10% quantitative improvement as to atleast one characteristic under at least one mode of application (a-e) ofthe composition. In some embodiments, results show at least a 25%quantitative improvement in at least one characteristic and/or astatistically significant improvement in at least two characteristics.

Example 27—Brassicaeae (Cruciferae)

Experiments are conducted to test effects of application of a microalgaebased composition to crop plants of the family Brassicaeae (Cruciferae).Application is done as in other examples herein, such that, in varioustreatments, (a) seeds are wetted or soaked in the composition; (b) thecomposition is applied to soil pre-germination; (c) the composition isapplied to soil post-germination; (d) the composition is appliedperiodically to soil during the growing season; and/or (e) thecomposition is applied to leaves of the plants once or periodicallyduring the growing season. Results are measures for appropriate plantcharacteristics including: seed germination rate, seed germination time,seedling emergence, seedling emergence time, seedling size, plant freshweight, plant dry weight, utilization, fruit production, leafproduction, leaf formation, thatch height, plant health, plantresistance to salt stress, plant resistance to heat stress, plantresistance to heavy-metal stress, plant resistance to drought,maturation time, yield, root length, root mass, color, insect damage,blossom end rot, softness, fruit quality, and sunburn. Results show atleast a 10% quantitative improvement as to at least one characteristicunder at least one mode of application (a-e) of the composition. In someembodiments, results show at least a 25% quantitative improvement in atleast one characteristic and/or a statistically significant improvementin at least two characteristics.

Example 28—Caricaceae

Experiments are conducted to test effects of application of a microalgaebased composition to crop plants of the family Caricaceae. Applicationis done as in other examples herein, such that, in various treatments,(a) seeds are wetted or soaked in the composition; (b) the compositionis applied to soil pre-germination; (c) the composition is applied tosoil post-germination; (d) the composition is applied periodically tosoil during the growing season; and/or (e) the composition is applied toleaves of the plants once or periodically during the growing season.Results are measures for appropriate plant characteristics including:seed germination rate, seed germination time, seedling emergence,seedling emergence time, seedling size, plant fresh weight, plant dryweight, utilization, fruit production, leaf production, leaf formation,thatch height, plant health, plant resistance to salt stress, plantresistance to heat stress, plant resistance to heavy-metal stress, plantresistance to drought, maturation time, yield, root length, root mass,color, insect damage, blossom end rot, softness, fruit quality, andsunburn. Results show at least a 10% quantitative improvement as to atleast one characteristic under at least one mode of application (a-e) ofthe composition. In some embodiments, results show at least a 25%quantitative improvement in at least one characteristic and/or astatistically significant improvement in at least two characteristics.

Example 29—Malvaceae

Experiments are conducted to test effects of application of a microalgaebased composition to crop plants of the family Malvaceae. Application isdone as in other examples herein, such that, in various treatments, (a)seeds are wetted or soaked in the composition; (b) the composition isapplied to soil pre-germination; (c) the composition is applied to soilpost-germination; (d) the composition is applied periodically to soilduring the growing season; and/or (e) the composition is applied toleaves of the plants once or periodically during the growing season.Results are measures for appropriate plant characteristics including:seed germination rate, seed germination time, seedling emergence,seedling emergence time, seedling size, plant fresh weight, plant dryweight, utilization, fruit production, leaf production, leaf formation,thatch height, plant health, plant resistance to salt stress, plantresistance to heat stress, plant resistance to heavy-metal stress, plantresistance to drought, maturation time, yield, root length, root mass,color, insect damage, blossom end rot, softness, fruit quality, andsunburn. Results show at least a 10% quantitative improvement as to atleast one characteristic under at least one mode of application (a-e) ofthe composition. In some embodiments, results show at least a 25%quantitative improvement in at least one characteristic and/or astatistically significant improvement in at least two characteristics.

Example 30—Sapindaceae

Experiments are conducted to test effects of application of a microalgaebased composition to crop plants of the family Sapindaceae. Applicationis done as in other examples herein, such that, in various treatments,(a) seeds are wetted or soaked in the composition; (b) the compositionis applied to soil pre-germination; (c) the composition is applied tosoil post-germination; (d) the composition is applied periodically tosoil during the growing season; and/or (e) the composition is applied toleaves of the plants once or periodically during the growing season.Results are measures for appropriate plant characteristics including:seed germination rate, seed germination time, seedling emergence,seedling emergence time, seedling size, plant fresh weight, plant dryweight, utilization, fruit production, leaf production, leaf formation,thatch height, plant health, plant resistance to salt stress, plantresistance to heat stress, plant resistance to heavy-metal stress, plantresistance to drought, maturation time, yield, root length, root mass,color, insect damage, blossom end rot, softness, fruit quality, andsunburn. Results show at least a 10% quantitative improvement as to atleast one characteristic under at least one mode of application (a-e) ofthe composition. In some embodiments, results show at least a 25%quantitative improvement in at least one characteristic and/or astatistically significant improvement in at least two characteristics.

Example 31—Anacardiaceae

Experiments are conducted to test effects of application of a microalgaebased composition to crop plants of the family Anacardiaceae.Application is done as in other examples herein, such that, in varioustreatments, (a) seeds are wetted or soaked in the composition; (b) thecomposition is applied to soil pre-germination; (c) the composition isapplied to soil post-germination; (d) the composition is appliedperiodically to soil during the growing season; and/or (e) thecomposition is applied to leaves of the plants once or periodicallyduring the growing season. Results are measures for appropriate plantcharacteristics including: seed germination rate, seed germination time,seedling emergence, seedling emergence time, seedling size, plant freshweight, plant dry weight, utilization, fruit production, leafproduction, leaf formation, thatch height, plant health, plantresistance to salt stress, plant resistance to heat stress, plantresistance to heavy-metal stress, plant resistance to drought,maturation time, yield, root length, root mass, color, insect damage,blossom end rot, softness, fruit quality, and sunburn. Results show atleast a 10% quantitative improvement as to at least one characteristicunder at least one mode of application (a-e) of the composition. In someembodiments, results show at least a 25% quantitative improvement in atleast one characteristic and/or a statistically significant improvementin at least two characteristics.

Example 32

Experiments were conducted to test variations in bacterial populationamong different batches of a low concentration mixotrophic Chlorellabased composition (PT Brown, PT Field, PT Fresh, PT Hydro, PT New and PTTexas). In all batches, dominant taxa included Paenibacillus, Bacillus,Lactobacillus, and Brevibacillus. Paenibacillus and Bacillus are knownto secrete a myriad of beneficial compounds in the rhizosphere of plants(phytohormones, nitrogenous compounds, antibiotics). They are also knownto mitigate pathogens. Lactobacillus is a fermentative lactic acidproducing bacterium used in the agricultural practice of silage.Brevibacillus is less known as a common plant growth promoting genus,however some literature exists demonstrating its ability to leach heavymetals from the rhizosphere. While some quantitative variability of eachbacterial population existed from batch to batch, these four sporulatingbacterial taxa were predominant. It is believed that the pasteurizationtreatment of the composition differentially suppressed othernon-sporulating taxa, which were found to be present but insignificantly lower amounts.

Example 33

Experiments were conducted to test the stability of variations inbatches of a microalgae Chlorella based composition at different storagetemperatures. Samples were tested monthly at various temperatures (2-5°C., 35° C., or 40° C.) for six months. Bacterial counts were determinedas well as levels of total nitrogen, phosphorous and potassium. Theresults of the experiments demonstrated that under different conditions,bacterial counts varied but nutrient levels remained essentially stable.

Example 34

Experiments were conducted to test different methods of preparation of amicroalgae Chlorella based composition (PT). Chlorella is capable ofheterotrophy (consuming an external carbon source), phototrophy(photosynthesis to convert CO₂ into a useable carbon source), and alsomixotrophy (simultaneously receiving nutrition/carbon via photosynthesisand also by consuming available external carbon sources). Compositionsof purely heterotrophic Chlorella, raised on two different carbonsources (either acetic acid or glucose), were compared with phototrophicChlorella and also with mixotrophic Chlorella, also grown in two batcheseach raised on either acetic acid or glucose as its carbon source. Theresults of the experiments are shown in FIG. 8. Mixotrophic culturesgrew most rapidly but also showed better results when applied to plantsas compared with other cultures.

Example 35

In one non-limiting example of mixotrophic culturing of Chlorella forthe described method of preparation of a composition for application toplants, the Chlorella is cultured in a BG-11 culture media or a mediaderived from BG-11 culture media (e.g., in which additional component(s)are added to the media and/or one or more elements of the media isincreased by 5%, 10%, 15%, 20%, 25%, 33%, 50%, or more over unmodifiedBG-11 media) for a culture length of 7-14 days in an open culturingvessel. The temperature can range from 20−30° C., or more, and the pHranges from 6.5-8.5. The dissolved oxygen concentration can range from0.1-4 mg/L. The culture receives acetic acid or acetate as a source oforganic carbon supplying carbon as energy source to the Chlorella cellsand also regulating the pH, and is supplied to the culture in a feedwith a concentration in the range of 10-90% by a pH auxostat system. Theculture receives natural sunlight (comprising photosynthetically activeradiation) as a source of energy. Mixing is provided by air spargingthrough an aerotube, and fluid propulsion by thrusters submerged in theliquid culture. Alternative organic carbon sources can include, forexample, any of: ammonium linoleate, arabinose, arginine, aspartic acid,butyric acid, cellulose, citric acid, ethanol, fructose, fatty acids,galactose, glucose, glycerol, glycine, lactic acid, lactose, maleicacid, maltose, mannose, methanol, molasses, peptone, plant basedhydrolyzate, proline, propionic acid, ribose, sacchrose, partial orcomplete hydrolysates of starch, sucrose, tartaric, TCA-cycle organicacids, thin stillage, urea, industrial waste solutions, yeast extract,any combination of the foregoing, or other organic carbon sources.

Example 36

In an alternative embodiment to the embodiment described in Example 40,aerators can be used as alternatives to spargers and submerged thrustersto provide both infusion of gases (e.g., oxygen) into the aqueousmicroalgae culture and turbulent mixing of the microalgae culture. Onenon-limiting example of an aerator for use as an alternative to thecombination of spargers and submerged thrusters is the Aire-02® Series275 Aspirator Aerator (Aeration Industries International, Chaska, Minn.USA). Such aerators include an electric motor drive above the culturemedia surface mounted on a float, a hollow shaft extending at an anglefrom above the culture media surface into the culture, and a propellerdisposed at end of the shaft which is submerged within the culturemedia. The motor is coupled to and drives the shaft and propeller. Thepropeller thrusts the aqueous culture media past a diffuser at the endof the shaft to induce a pressure differential in the hollow shaft,drawing air through intake holes in the shaft above the culture mediasurface down through the rotating hollow shaft and diffuser into themicroalgae culture. While the aerators contribute to the turbulentmixing and infusion of oxygen, the previously described devices andmethods of supplying nutrients, supplying organic carbon, andcontrolling pH can be used in conjunction with such aerators.

Example 37

In one non-limiting example of preparing the liquid composition with themixotrophic Chlorella based composition for application to plants, themixotrophic Chlorella based composition harvested from the culturingsystem is first held in a harvest tank before centrifuging the culture.Once the mixotrophic Chlorella culture is centrifuged, the centrifugedischarges the fraction rich in mixotrophic Chlorella whole cell solids,but also containing the accompanying constituents from the culturemedium, into a container at a temperature of about 30° C. Themixotrophic Chlorella based composition can continue (i.e., fresh) inthe process of preparing the liquid composition or be stored in afreezer and thawed at a later time (i.e., stored) for processing intothe liquid composition. When the mixotrophic Chlorella based compositionis stored in a freezer, the storage temperature is about −10° C. andabout 1-2 days are required for the composition to freeze. Once removedfrom the freezer, the stored mixotrophic Chlorella based composition isplaced outside to thaw for about 7 days. The fresh or stored mixotrophicChlorella based composition is then placed in a tank and heated to atemperature of about 60° C. for about 2 hours to begin thepasteurization process. The mixotrophic Chlorella based composition isthen diluted to a whole cells solids concentration of about 10-11% byweight and cooled to about 40° C. to complete the pasteurizationprocess. The pH of the mixotrophic Chlorella based composition is thenadjusted to a pH of about 4 by mixing in an effective amount ofphosphoric acid for stabilization purposes. About 0.3% potassium sorbateis then mixed with the mixotrophic Chlorella based composition forstabilization purposes. The resulting liquid composition is thentransferred to containers of a desired size stored at 3-5° C. untilshipped.

Example 38

Using QPCR (quantitative polymerase chain reaction) to analyze thebacteria population in a mixotrophic Chlorella culture beforepasteurization and after pasteurization, it was observed that theprofile of bacteria in the culture changes after pasteurization.Particularly, the post-pasteurization profile of bacteria includes ahigher proportion of spore forming bacteria and includes, but is notlimited to, Paenibacillus sp., Bacillus sp., Lactobacillus sp., andBrevibacillus sp as the dominant types of bacteria. Comparing theaerobic plate counts of a mixotrophic Chlorella culture beforepasteurization and after pasteurization, it was also observed that thetotal number of bacteria in the culture is lower after pasteurization.Combinations of temperature and time for the pasteurization process forthe times of 15, 30, 60, 120, 180, and 360 minutes, and 50, 6-0, 70, 80,and 90° C. were tested with a culture of mixotrophic Chlorella, and theresulting aerobic plate counts ranged from 7.58×106 CFU to as low as1.74×103 CFU. Storage temperature was also shown to vary the profile ofbacteria of a pasteurized culture of mixotrophic Chlorella, with samplesstored at temperatures of 2-4° C., 25° C., and 40° C. varying in theaerobic plate count numbers and type of dominant bacteria species overtime.

While the mixotrophic Chlorella cells are intact and viable (i.e.,physically fit to live, capable of further growth or cell division)after being harvested from the culture, the Chlorella cells resultingfrom the pasteurization process were confirmed to have intact cell wallsbut were not viable. Mixotrophic Chlorella cells resulting from thepasteurization process were observed under a microscope to determine thecondition of the cell walls after the being subjected to the heating andcooling of the process, and was visually confirmed that the Chlorellacell walls were intact and not broken open. For further investigation ofthe condition of the cell, a culture of live mixotrophic Chlorella cellsand the mixotrophic Chlorella cells resulting from the pasteurizationprocess were subjected to propidium iodide, an exclusion fluorescent dyethat labels DNA if the cell membrane is compromised, and visuallycompared under a microscope. The propidium iodide comparison showed thatthe Chlorella cells resulting from the pasteurization process containeda high amount of dyed DNA, resulting in the conclusion that themixotrophic Chlorella cell walls were intact but the cell membranes werecompromised. Thus, the permeability of the pasteurized Chlorella cellsdiffers from the permeability of a Chlorella cell with both an intactcell wall and cell membrane.

Additionally, a culture of live mixotrophic Chlorella cells and themixotrophic Chlorella cells resulting from the pasteurization processwere subjected to DAPI (4′,6-diamidino-2-phyenylindole)-DNA bindingfluorescent dye and visually compared under a microscope. The DAPI-DNAbinding dye comparison showed that the Chlorella cells resulting fromthe pasteurization process contained a greatly diminished amount ofviable DNA in the cells, resulting in the conclusion indicating that themixotrophic Chlorella cells are not viable after pasteurization. The twoDNA dying comparisons demonstrate that the pasteurization process hastransformed the structure and function of the Chlorella cells from thenatural state by changing: the cells from viable to non-viable, thecondition of the cell membrane, and the permeability of the cells.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Allprovided ranges of values are intended to include the end points of theranges, as well as values between the end points.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having,” “including,” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subjectmatter recited in the claims and/or aspects appended hereto as permittedby applicable law.

What is claimed is:
 1. A method of enhancing growth of a plantcomprising administering an effective amount of a liquid compositiontreatment comprising a culture of Chlorella to the plant, thecomposition comprising whole pasteurized Chlorella cells; whereinpresence of the whole pasteurized Chlorella cells in the compositionenhances the growth of the plant compared to a composition comprisingnon-pasteurized Chlorella cells, lacking the whole pasteurized Chlorellacells.
 2. The method of claim 1 wherein the effective amount is aconcentration in the range of 0.001-0.400% solids by weight.
 3. Themethod of claim 1 wherein the effective amount is a concentration in therange of 1-50 mL/gallon.
 4. The method claim 1 wherein the Chlorellacells are pasteurized at between 50-90° C.
 5. The method of claim 1wherein the Chlorella cells are pasteurized for a time between 15-360minutes.
 6. The method of claim 1 wherein the pH of the composition isadjusted to between 3-5.
 7. The method of claim 1 wherein the Chlorellacells are cultured in mixotrophic conditions.
 8. The method of claim 7wherein the Chlorella cells are cultured in non-axenic mixotrophicconditions, and wherein at least one species of sporulating bacterium ispresent in the non-axenic culture.
 9. The method of claim 8 wherein thebacterium is selected from: Paenibacillus sp., Bacillus sp.,Lactobacillus sp., and Brevibacillus sp.
 10. The method of claim 7wherein the Chlorella cells are cultured in axenic mixotrophicconditions.
 11. The method of claim 1 wherein the administering isselected from: soaking a seed in the composition prior to planting;contacting soil in an immediate vicinity of a planted seed with aneffective amount of the composition; contacting roots of a plant with aneffective amount of the composition hydroponically; and contacting aneffective amount of the composition to an accessible portion of theplant after emergence.
 12. The method of claim 11 wherein the liquidcomposition is administered at a rate in the range of 10-150 gallons peracre to soil or to emerged plants in soil.
 13. The method of claim 12wherein the liquid composition is administered at a rate in the range of10-50 gallons per acre by spraying onto plant foliage.
 14. The method ofclaim 12 wherein the liquid composition is administered at a rate in therange of 50-150 gallons per acre to the soil.
 15. The method of claim 11wherein the seed is soaked for 90-150 minutes.
 16. The method of claim 1wherein the liquid composition is administered to the soil by a systemselected from: a low volume irrigation system; a soil drenchapplication; and an aerial spraying system.
 17. The method of claim 16wherein the liquid composition is administered by spraying onto plantfoliage.
 18. The method of claim 1 wherein the plant is a member of aplant family selected from: Solanaceae, Fabaceae (Leguminosae), Poaceae,Roasaceae, Vitaceae, Brassicaeae, (Cruciferae), Caricaceae, Malvaceae,Sapindaceae, Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae,Lamiaceae, Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae(Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae,Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae,Rubiaceae, Papveraceae, Illiciaceae, Grossulariaceae, Myrtaceae,Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae),Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae,Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae,Piperaceae, and Proteaceae.
 19. The method of claim 1 wherein the wholeChlorella cells have not been subjected to a drying process.
 20. Themethod of claim 1 wherein the whole Chlorella cells have been subjectedto a drying process.
 21. The method of claim 1 wherein the liquidcomposition treatment further comprises at least one culture stabilizersuitable for plants.
 22. The method of claim 21 wherein the culturestabilizer is selected from: potassium sorbate, phosphoric acid,ascorbic acid, sodium benzoate, and any combination thereof.
 23. Themethod of claim 1 wherein the liquid composition treatment does notcontain an active ingredient for enhancing emergence or maturation otherthan the culture of whole Chlorella cells.
 24. The method of claim 1wherein the enhancement is determined by comparison of a treated plantwith a substantially identical untreated plant, and wherein aquantifiable difference of at least 10% is observed for at least oneplant characteristic.
 25. The method of claim 24 wherein the plantcharacteristic is selected from: seed germination rate, seed germinationtime, seedling emergence, seedling emergence time, seedling size, plantfresh weight, plant dry weight, utilization, fruit production, leafproduction, leaf formation, thatch height, plant health, plantresistance to salt stress, plant resistance to heat stress, plantresistance to heavy-metal stress, plant resistance to drought,maturation time, yield, root length, root mass, color, insect damage,blossom end rot, softness, fruit quality, and sunburn.
 26. The method ofclaim 25 wherein the characteristic is seedling emergence, and whereinthe number of plants emerged from the soil is increased by at least 10%compared to a substantially identical population of untreated plants.27. The method of claim 25 wherein the characteristic is maturation byleaf formation, and wherein the number of plants demonstratingmaturation by leaf formation is increased by at least 10% compared to asubstantially identical population of untreated plants.
 28. The methodof claim 1 wherein the liquid composition treatment further comprisesphytohormones selected from: abscisic acid, abscisic acid metabolites,cytokinins, auxins, gibberellins, and any combination thereof.
 29. Themethod of claim 1 wherein the liquid composition is pasteurized at aconcentration of 5-30% solids by weight of whole Chlorella cells.
 30. Amethod of enhancing growth of a plant comprising administering aneffective amount of a liquid composition treatment comprising a cultureof Chlorella to the plant, the composition comprising whole Chlorellacells pasteurized at a concentration of 5-30% whole Chlorella cells byweight; wherein presence of the whole pasteurized Chlorella cells in thecomposition enhances the growth of the plant compared to a compositioncomprising non-pasteurized Chlorella cells, lacking the wholepasteurized Chlorella cells.
 31. The method of claim 30 wherein theliquid composition is administered at a rate in the range of 10-150gallons per acre to soil or to emerged plants in soil.
 32. The method ofclaim 30 wherein the plant is a member of a plant family selected from:Solanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae, Vitaceae,Brassicaeae, (Cruciferae), Caricaceae, Malvaceae, Sapindaceae,Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae,Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae(Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae,Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae,Rubiaceae, Papveraceae, Illiciaceae, Grossulariaceae, Myrtaceae,Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae),Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae,Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae,Piperaceae, and Proteaceae.
 33. The method of claim 30 wherein theliquid composition treatment further comprises at least one culturestabilizer suitable for plants, wherein the culture stabilizer isselected from: potassium sorbate, phosphoric acid, ascorbic acid, sodiumbenzoate, and any combination thereof.
 34. The method of claim 30wherein the enhancement is determined by comparison of a treated plantwith a substantially identical untreated plant, and wherein aquantifiable difference of at least 10% is observed for at least oneplant characteristic, wherein the plant characteristic is selected from:seed germination rate, seed germination time, seedling emergence,seedling emergence time, seedling size, plant fresh weight, plant dryweight, utilization, fruit production, leaf production, leaf formation,thatch height, plant health, plant resistance to salt stress, plantresistance to heat stress, plant resistance to heavy-metal stress, plantresistance to drought, maturation time, yield, root length, root mass,color, insect damage, blossom end rot, softness, fruit quality, andsunburn.
 35. The method of claim 30 wherein the Chlorella cells arepasteurized at between 50-90° C. for a time between 15-360 minutes. 36.The method of claim 35 wherein the Chlorella cells are pasteurized in aculture having a concentration greater than 11% by weight of Chlorella,at between 55-65° C. for between 90-150 minutes, and wherein the cultureis then diluted to 10-11% Chlorella by weight and cooled to between35-45° C.
 37. The method of claim 30 wherein the pH of the compositionis adjusted to between 3.5-4.5.
 38. The method of claim 30 wherein theChlorella cells are cultured in mixotrophic conditions, the mixotrophicconditions comprising culturing the Chlorella cells in a suitable mediumfor a culture length of 7-14 days, at a temperature between 20-30° C.,at a pH between 6.5-8.5, and a dissolved oxygen concentration can rangebetween 0.1-4 mg/L.
 39. The method of claim 30 wherein the Chlorellacells are cultured in non-axenic mixotrophic conditions and at least onespecies of sporulating bacterium is present in the non-axenic culture,wherein the bacterium is selected from: Paenibacillus sp., Bacillus sp.,Lactobacillus sp., and Brevibacillus sp.
 40. The method of claim 30wherein the Chlorella cells are cultured in axenic mixotrophicconditions.